H3 Biomedicine Research Articles

Functional Oncogenomic and Immune Response Landscape for Genes Recurrently Mutated in Myeloma

For many genes that are recurrently mutated in patient-derived samples or cell lines from multiple myeloma (MM), the functional roles of these genes have remained incompletely understood. We have sought to address the functional implications of these genes through extensive CRISPR-Cas9-based functional genomics studies for loss-of-function (LOF, e.g. CRISPR-based gene editing) and gain of function (GOF, e.g. CRISPR-based gene activation) in MM cell lines in vitro and in vivo. We focused on 96 genes mutated in >2% of newly diagnosed MM patients in the CoMMpass (IA17) study (after excluding non-expressed genes [<1 FPKM in 95% of samples] and immunoglobulin/MHC genes) and specifically examined the performance of these genes in a series of in vitro genome-scale studies for CRISPR gene editing or activation (n=19 and n=5 MM lines, respectively); in vitro validation studies with subgenome-scale focused sgRNA libraries of individual sgRNAs; and in vivo focused subgenome-scale CRISPR screens (in 3 cell lines) in conventional subcutaneous (s.c.) xenograft or in bone marrow (BM)-like scaffolds with “humanized” mesenchymal BM stromal cell compartment (seeking a more translationally-relevant simulation of the BM microenvironment in MM patients).In these LOF and GOF functional studies, there were several examples of recurrently mutated genes which performed in vitro and in vivo in a manner consistent with prior knowledge on these genes as essential for MM survival/growth (e.g. NRAS or KRAS in MM lines harboring mutations for those genes, FGFR3 in t(4;14) lines) or, conversely, as tumor suppressor genes (e.g. TENT5C/FAM46C, TRAF3, TP53). Beyond these genes, which served as “positive controls”, other genes with notable functional roles identified in these studies include recurrently essential genes such as SETD2, the E3 ligases HUWE1 and HERC2, or the RNA binding protein genes RBMX and ELAVL1. Interestingly, however, the large majority of genes recurrently mutated in MM do not exhibit a recurrent role as either essential genes/drivers of MM cell growth or tumor suppressor genes in these in vitro or in vivo functional genomics studies. For instance, genes such as LTB, ATM, DUSP2, the phosphatases PTPRD, PTPRF, PTPRM do not exhibit recurrent functional role as major positive or negative regulators of MM cell growth in these studies, regardless of the mutational or copy number status of these genes in the cell lines tested.The fact that these observations were concordant in both in vitro and in vivo CRISPR studies suggests that the majority of genes recurrently mutated in MM patients do not seem to impact, either when lost or gained, the MM cell survival/proliferation in not only cell autonomous in vitro conditions, but also in the context of interaction with the local microenvironment of a “humanized” BM-like milieu. Given the immunocompromised nature of these in vivo systems, we also examined whether these recurrently mutated genes could influence the ability of MM cells to escape immune surveillance. However, in our in vitro genome-scale LOF or GOF CRISPR screens in three MM cell lines exposed to allogeneic donor-derived NK cells, perturbations of genes recurrently mutated in MM patient samples also did not emerge as “hits” associated with NK cell resistance.The fact that most recurrently mutated genes in MM did not elicit a clear phenotype in either loss- or gain-of-function studies with endpoints focusing on “cell fitness” (survival/proliferation) or in the context of exposure to immune effector cells such as NK cells, does not preclude alternative functional roles that may conceivably involve e.g. responses to other types of immune effector cells; cooperative interactions with other genes; roles in DNA repair (e.g. a plausible scenario in the case of ATM); impact on MM self-renewal; or potential role in early stages of myelomagenesis, but not in its late/advanced forms (which are generally considered to be represented by cell lines).More broadly, this study highlights the significance of identifying candidate drivers of MM cell biology by integrating conventional mutational analyses with functional characterization of the respective genes of interest, ideally through assessment of diverse endpoints related to both cell-autonomous and non-autonomous tumor cell behavior. DisclosuresTsherniak: Cedilla Therapeutics: Consultancy; Foghorn Therapeutics: Consultancy; The Center for Protein Degradation: Consultancy; SAB: Membership on an entity's Board of Directors or advisory committees. Licht: Epizyme: Research Funding. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Mitsiades: Nurix: Research Funding; EMD Serono: Research Funding; Novartis: Research Funding; BMS: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; Arch Oncology: Research Funding; Sanofi: Research Funding; H3 Biomedicine: Research Funding; FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees.

B Cell Transcriptional Coactivator POU2AF1 (BOB-1) Is an Early Transcription Factor Modulating the Protein Synthesis and Ribosomal Biogenesis in Multiple Myeloma: With Therapeutic Implication

Multiple Myeloma (MM) is a malignancy driven by numerous genetic and epigenetic alterations. Recurrent IgH translocations, somatic mutations and copy number abnormalities all contribute to myelomagenesis, however true drivers of the disease have not been well defined.To identify new targetable dependencies in MM, we generated high-quality active enhancer landscape using large cohort of primary patient myeloma cells (n=70), MM cell lines, and normal plasma cells. We integrated this data with an in-house curated atlas of 600+ active enhancer profile across a wide range of tumor types and normal tissues. Combining these data with gene expression and genetic dependency (CRISPR KO) enabled a multidimensional integration of how transcriptional regulation intersects with tumor specific dependencies. We identified that many of the specific and potent dependencies in MM are transcription factors, especially those establishing plasma cell identity. Among these, the POU2AF1 gene, which encodes the OCA-B/BOB-1, a B cell transcriptional coactivator protein, represented the most striking dependency in MM. Although BOB-1 is expressed throughout B-cell development, we found it to be highly expressed in CD138+ plasma cells from patients with precursor conditions (MGUS and SMM) as well as symptomatic MM compared to normal plasma cells.To functionally validate the role of BOB-1 in MM, we performed loss-of-function studies using shRNA, siRNAs and antisense GapMers specific for BOB-1 and observed a significant impact on MM cell viability and cell cycle arrest. Transcriptomic analysis upon BOB-1 depletion by RNA-sequencing revealed a small set of genes commonly modulated in all 3 MM cell lines tested including the plasma cell differentiation related transcription factor XBP1 and heme oxygenase (HMOX1). Importantly, we observed ribosome biogenesis, RNA polymerase 1A transcription and mRNA translation and elongation processes to be significantly enriched among genes modulated by BOB-1 depletion in MM cells. Bob1 KD resulted in a rapid and robust decrease in the level of transcription of rDNA by RNA polymerase I as determined by qRT-PCR quantification of pre-rRNA (47S). In addition, ChiP assay revealed decreased binding of RNA polymerase 1A to the 18S ribosomal DNA promoter region in BOB-1 depleted cells compared to control. These data indicate that BOB1 downregulation results in the suppression of RNA-polymerase I activity in MM cells.RNA Pol I-dependent transcription governs abundance of rRNA and directly regulates cellular translational and proliferative capacity. Since high protein load is a feature of MM, we evaluated the role of BOB-1 in the translational efficiency of MM cells. We observed that in MM cells compared to control cells, BOB-1 KD decreased, while its overexpression significantly enhanced de novo protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of BOB-1 perturbation on intracellular light chains (kappa or lambda) production. We observed changes in the intracellular abundance of the light chains with BOB-1 modulation in all MM cell lines tested. As a result of decreased protein production, BOB-1 depletion was associated with induction of resistance to proteasome inhibition suggesting that high expression of BOB-1 may be one the factors driving the exquisite sensitivity of MM cells to proteasome inhibitor. Interestingly, mass spectrometry analysis revealed BOB1 in a protein complex with mTOR, Raptor and mLST8 proteins which are members of mTORC1 complex which is also involved in ribosomal function and may suggest the mechanism of action of Bob-1 at molecular level.In conclusion, here we report BOB1 as a specific proximal dependency in MM cells with potential role in modulating the protein load/capacity balance and ribosomal biogenesis essential for MM cell protein production function and therefore their sensitivity to proteasome inhibition. DisclosuresShirasaki: FIMECS: Consultancy. Mitsiades: BMS: Research Funding; Nurix: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Abbvie: Research Funding; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Karyopharm: Research Funding; Sanofi: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria. Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria. Munshi: Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Pfizer: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Novartis: Consultancy.

Functional Genomic and Immune Response Characterization of PTEN Loss: Therapeutic Implications for Myeloma

PTEN (phosphatase and tensin homolog) is a known tumor suppressor gene (TSG) in solid tumors and hematologic malignancies, including multiple myeloma (MM), yet several aspects of its biology and clinical implications remain incompletely understood in MM. For instance, PTEN mutations/deletions are infrequent in newly diagnosed MM in the CoMMpass study (6 and 9 cases with heterozygous mutations or loss of one PTEN gene copy, respectively in 957 patients, IA16; no evidence of bi-allelic loss), and there is no enrichment for PTEN transcript suppression or biallelic PTEN loss at relapse. Moreover, while MM cell lines are considered to reflect advanced relapsed/refractory MM, nearly all are PTEN-proficient (with notable exceptions e.g. biallelic loss in OPM2). These considerations raise a question whether PTEN loss is indeed functionally associated with a competitive advantage for MM cells compared to their PTEN-proficient counterparts. In genome-scale CRISPR-Cas9 gene editing (knockout, KO) screens we observed that 16/19 MM cell lines exhibited CERES or CHRONOS scores >0.4, consistent with the proposed role of PTEN as a negative regulator of survival and proliferation: the 3 outlier cell lines included OPM2 (biallelic PTEN deletion) and 2 other lines (SKMM2 and AMO1) with no obvious genetic events suppressing PTEN function or causing constitutive activation of PI3K/Akt/mTOR signaling. We performed subgenome-scale CRISPR KO screens with focused libraries of single guide RNAs (sgRNAs) for several known (including PTEN) or candidate TSGs and control sgRNAs from MM and other tumors in vitro and confirmed that in vitro enrichment of PTEN KO in several PTEN-proficient (but not -deficient lines), consistent with the genome-scale CRISPR study results. These observations were further confirmed with in vitro competition assays where isogenic Cas9+ cells transduced with sgRNAs against PTEN or olfactory receptor (OR) genes (as DNA-cutting negative controls) were mixed, cultured for 4-12 weeks and DNA sequenced for the distribution of their sgRNAs, and determined a significant relative enrichment of PTEN KOs compared to their OR KO counterparts. Importantly, when cells harboring focused sub-genome scale sgRNA library were implanted in vivo in a bicalcium phosphate scaffold-based bone marrow (BM)-like model with a “humanized” mesenchymal bone stromal cell compartment, we observed that CRISPR KO of PTEN represented the top enriched gene perturbation in the PTEN-proficient MM cell lines MM.1S and KMS11 (not the AMO1 line) in vivo, consistent with the in vitro results. Collectively, these observations indicate that while PTEN deficiency is infrequent in newly diagnosed MM patient samples and in preclinical cell line models that reflect advanced disease, PTEN KO is associated with major “fitness” advantage for MM cells in preclinical in vitro and in vivo xenograft models, i.e. systems in which tumor cell “fitness” does not account for potential immunogenicity of human tumor cells. We hypothesized that PTEN KO remain highly immunogenic which curtails their “fitness” advantage: indeed, we observed in a series of CRISPR KO screens in PTEN-proficient cell lines from MM, other hematologic malignancies or solid tumors, PTEN loss was not associated with increased resistance to allogeneic donor-derived NK cells (in-house studies) or T-cell treatment (e.g. 41T, RENCA cells; GSE149933). In fact, for some MM cell lines (e.g. MM1S, KMS11), PTEN KO led to more pronounced sensitivity to NK cells (compared to control OR KOs) in genome-scale or focused CRISPR screens, and in validation experiments with individual sgRNAs for PTEN (vs. OR gene sgRNAs). While our functional genomic studies document that PTEN loss confers a generalizable “fitness advantage” for MM lines in vitro and in immunocompromised in vivo models, the limited, if any, association of advanced MM with PTEN loss raises intriguing questions about the potentially complex role of PTEN as a TSG. While alternative hypotheses are plausible, our results point to the immune responses of PTEN-deficient tumor cells as an additional factor that can reconcile our integrated observations in molecular annotated clinical samples and preclinical functional genomic studies. DisclosuresTsherniak: Cedilla Therapeutics: Consultancy; Foghorn Therapeutics: Consultancy; The Center for Protein Degradation: Consultancy; SAB: Membership on an entity's Board of Directors or advisory committees. Licht: Epizyme: Research Funding. Boise: Abbvie: Consultancy; AstraZeneca: Consultancy, Research Funding. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; EMD Serono: Research Funding; Abbvie: Research Funding; Nurix: Research Funding; Janssen/Johnson & Johnson: Research Funding; BMS: Research Funding; Ionis Pharmaceuticals: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; TEVA: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding.

Functional Genomic Landscape of Natural Killer Cell Evasion in Multiple Myeloma

Natural killer (NK) cell-based therapies are considered promising future approaches for multiple myeloma (MM) treatment, but immune evasion mechanisms are poorly understood. To determine the mechanisms regulating MM cell response to NK cells, we performed genome-wide (GW) and targeted CRISPR screens in MM cell lines. To further investigate the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed additional pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform and integrated these findings with data from phenotypic assessment of NK cell response of pooled “DNA-barcoded” cell line including 15 MM cell lines (PRISM platform) and molecular profiling of MM patient samples.Our loss-of-function (LOF) and gain-of-function (GOF) GW-CRISPR screens were performed in the MM1.S, LP1, KMS11 MM lines treated with ex vivo expanded NK cells vs untreated control. The top LOF hits were validated using a focused library of ~600 genes. As expected, LOF of class I HLA /antigen presentation machinery genes, transcriptional regulators of HLA and IFNg pathway genes sensitized MM cells to NK cells, confirming that these pathways represent prominent suppressors of NK cell killing. Moreover, LOF of death receptors or downstream effectors was associated with NK resistance; while LOF of the negative regulators of death receptor signaling (e.g. CFLAR, and XIAP) sensitized to NK cell killing.To mechanistically dissect the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed scRNAseq using the CROP-seq platform. Pools of MM1.S and LP1 expressing single-guide (sg)RNAs targeting 31 select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection, differential gene-expression analysis and patient data correlation.The single cell transcriptional profiling of each CRISPR-based LOF for genes of interest documented that disruption of TRAF2, NFKBIA or NFKBIB enhanced NF-kB signaling and was associated with increased expression of the death receptor FAS, a key mediator of NK cell sensitivity (Figure), and changes in expression of BIRC3, CD70, and CXCL10(the latter may further increase immune reactivity through recruitment of T and NK cells). Interestingly, in our PRISM data, high transcriptional NF-kB signatures and/or presence of TRAF3 mutations (an activator of NF-kB signaling) correlated with decreased NK cell response. Among the individual genes most highly correlated with NK cell resistance was CFLAR, a known NF-kB target gene, and negative regulator of death receptor-mediated apoptosis. In contrast, PTEN and NLRC5 mutations were associated with enhanced NK cell sensitivity in several MM lines tested, consistent with their depletion in the GW LOF CRISPR screens and the HLA I regulatory function of NLRC5.We correlated our findings with patient-derived data from the CoMMpass study. Mutations of NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes, consistent with our CROP-seq data. NFKBIA mutations were also linked to reduced HLA-E expression in both MM patient and CROP-seq data, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. NLRC5 mutations were associated with lower HLA-E expression consistent with CROP-seq data, suggesting that, although rare, NLRC5mutations may represent a more NK-sensitive MM subset. TRAF3 alterations occurred both in a distinct TRAF3-altered cluster and in a subset of CoMMpass patients with WHSC1 translocations, consistent with the observations in our PRISM data set. CFLAR expression was enriched in patients with TRAF3 alterations belonging to both of these groups. The aggregate of molecular data from MM patient samples and PRISM or CRISPR-based functional studies preclinically raise the possibility that TRAF3 and WHSC1/t(4;14) alterations may contribute, at least in part through CFLAR, to decreased NK cell response in MM cells.Our data illuminate the complex mechanisms of response to NK cells in MM, highlighting the different effects of distinct molecularly defined subgroups of MM tumor cells with increased susceptibility to NK cell treatment, underlining the potential of such studies as a blueprint for identification of biomarkers individualized use of NK cell-based therapies in MM. [Display omitted] DisclosuresMustjoki: Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Janpix: Research Funding. Mitsiades: H3 Biomedicine: Research Funding; FIMECS: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Nurix: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; BMS: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Arch Oncology: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals: Consultancy, Honoraria.

Multi-Recombinase Mouse Models of Flt3-Driven Leukemia Identifies Distinct Trajectories of Mutational Cooperativity and Leukemic Transformation

Genomic studies in acute myeloid leukemia (AML) have generated a near complete catalogue of genes mutated at varying frequencies both across patients and in individual leukemias. The high variability of mutation burden within a given leukemia is suggestive of a stepwise evolutionary process composed of early, clonal, mutations and subsequent subclonal events. The receptor tyrosine kinase, FLT3, is the most commonly mutated gene in AML, with mutations frequently manifesting as internal tandem duplications (ITDs) in the juxtamembrane domain leading to constitutive kinase activation. Although FLT3 is commonly a subclonal mutational event, FLT3 ITD mutations portend a poor prognosis particularly when combined with DNMT3A and NPM1, earlier mutations that drive clonal expansion. Notwithstanding its role as a subclonal driver, previous preclinical FLT3 models have utilized retroviral overexpression or germline mutant expression at the endogenous locus precluding accurate temporal modeling of disease. These efforts have prohibited evaluation of FLT3 mutational acquisition in the context observed in AML patients.Here, we report the development of an endogenously targeted, Flp inducible, Flt3 ITD mouse allele which can be somatically activated subsequent to cooperating disease alleles. When activated with a tamoxifen inducible FlpoER, Flt3 mutant mice developed rapid leukocytosis peaking at 4-6 weeks post activation and resolving by 8-10 weeks, a finding not previously observed in constitutive models. This leukocytosis was disproportionately monocytic and accompanied by pronounced anemia and thrombocytopenia. Long term, these mice develop a myeloproliferative disease , reminiscent of previously reported constitutive alleles. In competitive transplantation studies, Flt3 mutant cells initiated disease and outcompeted wild-type cells. Despite this competitive advantage, disease was incapable of transplanting into secondary recipients. We further observed a non-cell autonomous depletion of SLAM+ LSKs suggesting the Flt3 mutant cells cannot propagate disease in self-renewing stem cells.To evaluate how this allele influenced leukemic evolution we crossed this Flt3 ITD allele to a Flp inducible Npm1 c mouse where a pulse of tamoxifen simultaneously activated both alleles. The combination of mutant Npm1 and Flt3 resulted in progressive leukocytosis which did not resolve. Within 6 weeks of mutational activation, these mice developed a lethal AML with robust anemia, thrombocytopenia, leukocytosis and expanded cKIT+ blasts in the blood. RNA-sequencing and immunophenotyping by CyTOF revealed distinct patterns of differentiation, gene-expression and downstream signaling.In an effort to model sequential mutational acquisition, we crossed the Flp Flt3 ITD allele to a Cre-inducible Dnmt3a R878H. Cre mRNA was electroporated into lineage negative bone marrow cells to activate the Dnmt3a R878H allele and transplanted into lethally irradiated recipients. Four weeks post engraftment, Flt3 ITD was activated with a pulse of tamoxifen. In contrast to the Flt3-Npm1 model, we observed an increase and subsequent decrease in WBC similar to the kinetics observed in Flt3 ITD only mice. However, by 20 weeks we observed a robust and consistent increase in WBC accompanied by an emergence of cKIT+ cells in the blood. Histopathology indicated that >50% of mice expressing both alleles in sequence developed AML marked by increased blasts in the marrow, with moderate anemia and thrombocytopenia compared to the Flt3-Npm1 models. Critically, in contrast to Flt3 ITD only mice, acquisition of the Flt3 ITD in Npm1 or Dnmt3a mutant HPSCs induced fully transplantable AML with immunophenotypic characteristics seen in human AML with these same genotypes. Collectively these results demonstrate that different co-occurring mutations are capable of transforming Flt3 ITD mutant cells, albeit with distinct latencies and mechanisms of cooperativity.In summary, our studies utilizing novel multi-recombinase models of leukemogenesis reveal new insights into the early phase of oncogene activation, and how cooperating alleles influence this response. This inducible Flt3 ITD allele represents a significant advance in modeling clonal evolution in myeloid malignancies and provides a critical isogenic platform for preclinical development of novel leukemia therapeutic regimens. [Display omitted] DisclosuresBowman: Mission Bio: Honoraria, Speakers Bureau. Xiao: Stemline Therapeutics: Research Funding. Miles: Mission Bio: Honoraria, Speakers Bureau. Trowbridge: Fate Therapeutics: Patents & Royalties; H3 Biomedicine: Research Funding. Levine: Amgen: Honoraria; Lilly: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Ajax: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Zentalis: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Incyte: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Treatment Patterns in Patients with Chronic Myeloid Leukemia in Chronic Phase in the Third Line of TKI Therapy and Beyond Based on Real-World Evidence

Introduction: Tyrosine kinase inhibitors (TKIs) are the standard of care for patients with chronic myeloid leukemia in chronic phase (CML-CP). The advent of TKIs has led to improvements in survival, with a life expectancy nearly matching that of the general population. However, TKI failure rates increase with subsequent lines of therapy, leading to worse overall survival in patients in later lines of therapy. There are 5 approved TKIs (imatinib [IMA], bosutinib [BOS], dasatinib [DAS], nilotinib [NIL], and ponatinib [PON]) that may be used based on patient characteristics. IMA is frequently used in first-line (1L) therapy. Second-generation TKIs (BOS, DAS, and NIL) can be used in the 1L or second line (2L) depending on choice of 1L therapy. Use of PON is reserved mostly for patients with T315I mutations due to its adverse event profile. However, little guidance exists concerning which TKI to use in the third line and beyond (3L+). Here we present the results of a real-world evidence study of treatment patterns of TKIs in patients with CML-CP initiating 3L+ TKI therapy.Methods: This is a noninterventional, descriptive cohort study based on secondary use of data from 3 existing CML registries (Czech INFINITY, Dutch PHAROS, and Swedish CML). The observation period was from January 1, 2008, until the last available date of follow-up for each registry (6-12 years depending on the registry). Patients aged ≥18 years with CML-CP initiating a 3L TKI after registry enrollment were included in this analysis. Exposure was defined by TKI treatment with IMA, DAS, NIL, BOS, and PON. Descriptive statistics were used to describe patient demographics and treatment duration. Alluvial diagrams were used to describe and represent all treatment patterns.Results: Of those patients that had started a 2L TKI in the INFINITY, PHAROS, and Swedish CML registries, 109 (28.1%), 51 (35.7%), and 182 (12.8%) patients began 3L TKI therapy, with 105, 48, and 172 eligible patients, respectively, included in this study. Median age at diagnosis was 54.0, 61.0, and 59.6 years, respectively (Table 1). There were more men than women across all 3 registries (51.4%, 58.3%, and 50.6% vs 48.6%, 41.7%, and 49.4%, respectively). No agreement in treatment sequencing was seen across the 3 registries. However, across all 3 registries, the most frequently prescribed TKI in the 3L+ setting was NIL (52.4%, 59.6%, and 45.4%, respectively), followed by DAS (38.1%, 17.0%, and 32.6%, respectively). BOS (11.4%, 0%, and 27.9%, respectively), and PON (13.3%, 17.0%, and 11.1%, respectively), which were prescribed at varying frequencies across the registries. The alluvial plot in the Figure presents the sequence of TKI treatments for patients starting a 2L TKI. Median treatment duration in the 3L was longest for patients receiving NIL (27.5, 13.1, and 45.0 months, respectively) and DAS (26.6, 10.0,9.9 and 40.9 months, respectively), except in PHAROS, in which PON resulted in a longer treatment duration than DAS (6.0, 11.4, and 7.2 months, respectively).In INFINITY, rates of resistance (R) and intolerance (I) in patients discontinuing therapy with BOS, DAS, and NIL were 28.6% and 42.9%, 61.1% and 38.9%, and 48.0% and 36.0%, respectively. In PHAROS, 100.0% of patients discontinued DAS and IMA in the 3L due to I, while R and I were reasons for discontinuation in 16.7% and 83.3% of patients discontinuing NIL and 66.7% and 33.3% of patients discontinuing PON, respectively. The Swedish CML register reported that 15.8%, 9.1%, and 33.3% of patients discontinued DAS, NIL, and PON, respectively, in the 3L+ due to I (Table 2).Conclusions: Based on these real-world treatment patterns (which may provide insights into TKI access in various settings), patients in the 3L were most frequently treated with the second-generation TKIs NIL and DAS, with a smaller proportion treated with BOS and PON. However, no common agreement in sequencing of TKI treatment was seen across the 3 registries. Duration of treatment was longest in patients receiving NIL and DAS. Treatment discontinuation occurred frequently due to both R to and I of the 3L TKI used. Between 12.8% and 35.7% of patients that started a 2L TKI required 3L+ therapy, and therefore additional treatment options that are efficacious and well tolerated are needed for these patients.This study was a research collaboration between Novartis and the 3 registries and was funded by Novartis. [Display omitted] DisclosuresDahlen: Novartis: Research Funding. Ferreira: Novartis: Consultancy; Roche: Consultancy; MerckGroup: Consultancy; Research institute for the study of genetic and malignant disorders in children.: Consultancy. Westerweel: Pfizer: Consultancy; BMS / Celgene: Consultancy; Incyte: Consultancy; Novartis: Research Funding. Mayer: Principia: Research Funding. Sahmoud: Novartis: Current Employment; H3 Biomedicine: Consultancy; Context Therapeutics: Consultancy. Wormser: Roche: Current equity holder in publicly-traded company; Novartis: Current Employment, Current equity holder in publicly-traded company. Žácková: Novartis: Speakers Bureau; Angelini: Consultancy, Speakers Bureau.

Aberrant CDK7 Activity Drives the Cell Cycle and Transcriptional Dysregulation to Support Multiple Myeloma Growth: An Attractive Molecular Vulnerability

Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models.Gene expression analysis after CDK7 inhibition in several MM cell lines revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs.To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented.In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. DisclosuresShirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

Spliceosomal Dysfunction Is a Critical Mediator of IDH2 Mutant Leukemogenesis

Mutations affecting RNA splicing factors are commonly found in leukemias and mutations in SF3B1, SRSF2, and U2AF1 impart specific changes to global splicing. We analyzed RNA-seq data from acute myeloid leukemia (AML) patients from The Cancer Genome Atlas (TCGA) to evaluate for spliceosomal alterations characteristic of mutant SRSF2, SF3B1, and U2AF1 . Although only 1 patient with an SRSF2 mutation was reported in the TCGA AML publication, we identified 19 additional patients to have the characteristic splicing changes of mutant SRSF2 . Mutational analysis of the RNA-seq data identified the SRSF2 proline 95 hotspot mutation in each of these 19 patients (19/178, 11%), making SRSF2 amongst the most commonly mutated genes in the AML TCGA dataset. Interestingly, 47% of SRSF2 mutant patients also had a co-existing IDH2 mutation (and conversely, 50% of IDH2 mutant patients had a co-existing SRSF2 mutation). These data are similar to recent observation that SRSF2 mutations were the most common accompanying mutation in IDH2 mutant myeloid leukemia patients treated on the phase I clinical trial of AG-221.We next sought to understand the functional contribution of SRSF2 mutations to co-existing IDH2 mutations. Overexpression of IDH2 R140Q and IDH2 R172K in Vav -cre Srsf2 P95H or Vav- cre Srsf2 wildtype(WT) backgrounds revealed a clear collaborative effect of mutant IDH2 and mutant Srsf2 (Figure A-B). As early as 4 weeks post-transplant, the peripheral blood of Srsf2 mutant mice transplanted with IDH2 R140Q or IDH2 R172K forms had a substantially greater percentage of GFP+ cells than seen in an Srsf2 WT background. Moreover, these mice exhibited significant myeloid skewing, macrocytic anemia, and thrombocytopenia of greater magnitude than seen with mutant IDH2 expressed in the SRSF2 WT background. IDH2 / Srsf2 double-mutant mice developed a lethal, mixed myelodysplastic/myeloproliferative neoplasm that was serially transplantable (Figure B). Effects from overexpression of mutant IDH2 were recapitulated in mice expressing Idh2 R140Q and Srsf2 P95H from endogenous loci(Mx1 -cre Idh2R140Q/+Srsf2P95H/+ double knock-in mice) (Figure C), providing the first model where spliceosomal gene mutations promote leukemogenesis in vivo .Given prior data identifying inhibition of TET2 as a mediator of IDH2 -mutant leukemogenesis, we evaluated if loss of TET2 might promote transformation of SRSF2 mutant cells. However, deletion of TET2 in an SRSF2 mutant background (Mx1 -cre Tet2fl/flSrsf2P95H/+) was insufficient to promote transformation (Figure D). We next analyzed RNA-seq data from the TCGA, Leucegene, and our own unpublished AML cohort using a novel analysis method to detect unannotated as well as complex splicing events that might explain the basis for the collaboration of mutant IDH2 and SRSF2 . This revealed that IDH2/SRSF2 double-mutant AML had increased aberrant splicing events compared to SRSF2 single-mutant AML and also that IDH2 -mutations are associated with a small but reproducible change in RNA splicing (Figure E). The vast majority of splicing changes affected by SRSF2 and/or IDH2 mutations were a change in cassette exon splicing with SRSF2 mutations promoting inclusion of C-rich containing mRNA sequences, regardless of the presence of concomitant IDH2 mutations (Figure F). One of the most robust mis-splicing events in IDH2/SRSF2 mutant AML was an intron retention event in INTS3 resulting in its reduced mRNA and protein expression (Figure G-H). INTS3 encodes a component of the integrator complex that participates in small nuclear RNA (snRNA) processing. Consistent with this, IDH2 / SRSF2 double-mutant cells exhibited alterations in processing of U2 and U4 snRNAs similar to that seen with direct INTS3 downregulation. Biologically, attenuation of INTS3 expression caused a blockade of myeloid differentiation in HL-60 cells upon ATRA treatment, an effect which was further enhanced in an IDH2 mutant background.These data uncover an important role for spliceosomal gene mutations in the pathogenesis of IDH2 mutant leukemogenesis and identify perturbations in the integrator complex as a novel mediator driving the transformation of IDH2 and SRSF2 mutant cells. The frequent coexistence of IDH2 and SRSF2 mutations underscores the enormous therapeutic potential for modulation of splicing in the ~50% of IDH2 -mutant leukemia patients who also harbor a spliceosomal gene mutation. [Display omitted] DisclosuresDe Botton:Servier: Honoraria; Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Stein:Pfizer: Consultancy, Other: Travel expenses; Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding; GSK: Other: Advisory Board, Research Funding; Constellation Pharma: Research Funding. Peng:H3 Biomedicine Inc.: Employment. Buonamici:H3 Biomedicine Inc.: Employment. Palacino:H3 Biomedicine Inc.: Employment. Smith:H3 Biomedicine Inc.: Employment. Thompson:Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Levine:Roche: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding.

Splicing Modulation Perturbs Key Survival Pathways and Sensitizes Chronic Lymphocytic Leukemia to Venetoclax Treatment

Splicing Modulation Perturbs Key Survival Pathways and Sensitizes Chronic Lymphocytic Leukemia to Venetoclax Treatment

Dynamic BH3 Profiling to Assess the Effects of Novel Agents on Anti-Apoptotic Protein Dependence of CLL Cells

IntroductionOur group previously developed dynamic BH3 profiling (DBP) (Montero el al, 2015), which measures early changes in net pro-apoptotic signaling of mitochondria in response to various drug combinations. The degree of change in the propensity to undergo apoptosis is known as “delta priming”. We recently used DBP to demonstrate that BTK inhibition with ibrutinib leads to increased BCL-2 dependence and enhanced sensitivity to the BCL-2 inhibitor venetoclax in CLL cells (Deng et al., 2017). The effects of other novel agents on BCL-2 dependency in CLL cells relative to other anti-apoptotic family members such as MCL-1 and BCL-XL have yet to be systematically explored.Pathways that are recurrently mutated in CLL represent a logical next area in which to explore the potential of DBP to study the effects of novel agents on the BCL-2 family. One such gene is splicing factor 3B subunit 1 (SF3B1), which is recurrently mutated in a subset of patients with CLL, and confers an adverse prognosis. Here, we treated CLL cells ex vivo with a panel of novel agents including the splice modulator E7107, and utilized DBP to evaluate the functional effects of drug treatments on BCL-2 family proteins.MethodsMononuclear cells were isolated from 13 CLL patients and treated ex vivo with E7107 (H3 Biomedicine) and/or one of several other drugs (all obtained commercially). Cells were co-cultured with the human stroma NKTert cell line (1:10 ratio) with 20-hour drug treatments. DBP was performed as previously reported by measuring the release of cytochrome C (cyto-C) in gently permeabilized CLL cells in response to BH3-only peptides using a BD FACS Fortessa. Cyto-C release in response to BAD BH3 peptide was used as a measure of BCL-2 dependence, while cyto-C release in response to HRK and MS-1 BH3 peptide was used as a measure of BCL-XL and MCL-1 dependence, respectively. Western blot analyses were performed by standard protocols with signals captured by an LAS 4000 imager. Statistical analyses were by Welch t-test and one-sample t-test, with a two-tailed nominal p≤0.05 considered as significant.ResultsAs an initial screen, we treated cells from two different CLL patients in duplicate with a panel of 13 drugs and found that 5 agents resulted in increased BCL-2 dependence (defined as at least 35% cyto C release in both patients) (Fig. 1). These included E7107, ibrutinib, fludarabine, LY2784544 (JAK2 inhibitor), and AZD8055 (AKT inhibitor). As the greatest increase in BCL-2 dependence was observed with E7107, we next explored its effects on 11 additional CLL patient samples (6 SF3B1mut and 5 SF3B1wt). Significantly higher baseline BCL-2 dependence was observed in untreated SF3B1mut cells compared to SF3B1wt (p=0.02) (Fig. 2A). Treatment with E7107 alone or in combination with venetoclax increased BCL-2 dependence of CLL cells irrespective of SF3B1 mutation status (p values between 0.01-0.02). Venetoclax treatment alone had no effect on BCL-2 dependence in either group (p=0.39, p=0.68). In all samples, E7107 treatment made CLL cells significantly more BCL-2 dependent (BAD BH3 peptide, p=0.0009) and less MCL-1 dependent (MS-1 BH3 peptide, p<0.0001), but did not affect BCL-XL dependence (HRK BH3 peptide, p=0.11) (Fig. 2B). Consistent with these findings, CLL cells treated with E7107 had uniformly decreased MCL-1 protein expression on Western blot compared to untreated samples across all patient samples.ConclusionWe demonstrate the feasibility of using DBP to examine the effects of a panel of novel agents on BCL-2 family anti-apoptotic protein dependence of CLL cells. We identified several candidate drugs to explore in future studies, including the splice modulator E7107, which consistently increased BCL-2 dependence while reciprocally decreasing MCL-1 dependence. These preliminary results support the exploration of DBP in a high throughput format on a larger panel of drugs across more patient samples, with a goal of prioritizing the combinations that appear most promising to explore in clinical trials, to develop more personalized treatment for patients with CLL. [Display omitted] DisclosuresValentin:Abbvie: Other: Travel reimbursement; Roche: Other: Travel reimbursement. Buonamici:H3 Biomedicine, Inc.: Employment. Neuberg:Synta Pharmaceuticals: Other: Stock shares. Wu:Neon Therapeutics: Consultancy. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Davids:Merck: Consultancy; InCyte: Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Research Funding; Celgene: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy.

Preliminary Results from an Ongoing Phase 1/2 Study of INCB057643, a Bromodomain and Extraterminal (BET) Protein Inhibitor, in Patients (pts) with Advanced Malignancies

Preliminary Results from an Ongoing Phase 1/2 Study of INCB057643, a Bromodomain and Extraterminal (BET) Protein Inhibitor, in Patients (pts) with Advanced Malignancies

Integration of Functional Dependencies and Molecular Alterations of Myeloma Cells

During the last two decades, multiple myeloma (MM) cell lines and patient-derived samples have been extensively profiled for alterations in their genome, transcriptome, epigenome, and proteome, with the anticipation that molecules with the most recurrent or pronounced dysregulation (e.g. compared to nonmalignant plasma cells) could represent attractive novel therapeutic targets. Functional genomics platforms, including CRISPR/Cas9 systems, provide direct quantitative information on the impact of gene perturbation on tumor cell survival and proliferation, and we evaluated the information provided from such genome-wide studies on MM cell lines in vitro to examine how much overlap exists in the landscapes of vulnerabilities vs. molecular alterations in MM cells. The comparison of CRISPR essentiality screens in MM cell lines (n=10) vs. 300+ non-MM cell lines from solid tumors and hematologic malignancies allowed us to identify 40+ genes representing preferential or more pronounced/recurrent dependencies for MM cells vs. non-MM cells. These genes prominently featured transcription factors (e.g. IRF4, PRDM1, MAF, IKZF1, IKZF3, NFKB pathway members ), epigenetic regulators (e.g. EP300), and kinases (e.g. PIM2, IKBKB). Our integrated molecular evaluation of these genes examined a series of molecular alterations, including transcript overexpression in MM cell lines (or patient derived samples) vs. normal plasma cells, or in relapsed/refractory vs. earlier stages of disease; mutational status; transcript levels correlating with shorter progression free or overall survival across multiple molecularly-annotated clinical trial data sets in MM; and extent of open chromatin (based on H3K27Ac chromatin marks) within or proximal to each gene in MM cell lines. While some of the candidate MM-preferential essential genes exhibited at least one of the aforementioned molecular alterations, the large majority of these genes either harbored no such alterations or were not ranked in the top 50-100 genes in terms of magnitude or frequency of these alterations. Notably <10% of MM newly diagnosed MM patients harbor mutations in any of these genes; and only 4 genes had mutations in >1 MM cell line examined in our screen. While some MM-preferential dependencies genes (e.g. IRF4, PRDM1, MAF) have higher transcript levels in MM vs. non-MM cell lines, most of the 300+ genes that fall in the latter category are not selectively essential for MM cells. We extended our study to genes reported to have recurrent non-synonymous mutations in MM: among the 50+ genes with >3% mutation rate in newly diagnosed MM patient-derived cells, only 5 genes appeared as a dependency in 2 or more MM cell lines tested and only 1 gene had CRISPR data compatible with a candidate role as negative regulator of survival/proliferation. Focusing the analysis on specific categories of mutations did not alter this conclusion. While we observed several examples of MM cells lines in which known oncogenic events (e.g. mutations in KRAS or NRAS) were associated with dependency on the respective genes, we also observed other examples in which dependency on such genes was identified even in their wild-type state and without identifiable loss-of-function events in known negative regulators. These observations collectively support the notion that functional genomics approaches can identify promising candidate therapeutic targets, which may not be readily identifiable based on established annotations of molecular alterations in the genome, transcriptome, or epigenome of tumor cells. DisclosuresEbert:Genoptix: Patents & Royalties; H3 Biomedicine: Consultancy; GRAIL: Consultancy; Celgene: Research Funding. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Ono: Research Funding; Takeda: Other: Employment of family member; Novartis: Research Funding; Abbvie: Research Funding.

Prognostic Role of Gene Mutations in Chronic Myelomonocytic Leukemia (CMML) Patients Treated with Hypomethylating Agents. a Report on 183 Patients

Context: Somatic gene mutations contribute to the heterogeneous prognosis of CMML. The hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DAC) are active in CMML. The specific prognostic value of recurrent gene mutations in CMML treated with HMA has never been reported. Methods: We analyzed a multi-institution retrospective cohort of 183 CMML patients (pts) treated with either AZA or DAC with information on recurrent gene mutations. Results: CMML pts with a median age of 72 years (WHO 2016: CMML-0/1/2 in 37%, 27% and 36% respectively [resp]) were treated with AZA (n=76) or DAC (n=107) for a median of 7 cycles (InterQuartile Range [IQR]: 4 - 15). Median WBC was 15.1 x109/L, cytogenetic risk according to CPSS (Such et al, Haematologica 2011) was low, int and high in 71%, 12% and 17% of pts, resp. CPSS risk (Such et al, Blood 2013) was low, int-1, int-2 and high in 14%, 23%, 49% and 14%, resp. GFM risk (Itzykson et al, JCO 2013) was low, int and high in 33%, 35% and 32%, resp. The Overall Response Rate (ORR) to HMA was 54%, including 17% of complete responses (CR), without significant differences between AZA and DAC. With a median follow-up of 36.7 months, median overall (OS) and AML-free (AMLFS) survival were 24.4 and 19.2 months, resp. Nineteen (10%) pts were transplanted after HMA and censoring at transplant did not affect our results. Compared to AZA pts, DAC pts had higher WBC (p=0.01) and non-significant trends towards lower platelet count (p=0.07) and poorer cytogenetic risk (p=0.08), with a non-significant trend to a poorer OS (p=0.11). Propensity matching on previous treatment with chemotherapy, WBC, platelets count, peripheral blasts count, cytogenetic risk and ASXL1 status at HMA onset confirmed the lack of OS benefit with AZA in the 144 patients (79%) that could be successfully matched (Hazard Ratio [HR]=1.31, p=0.4). All further analyses were nevertheless stratified on HMA. Molecular data was obtained from Sanger sequencing (n=74) or NGS (n=109). The molecular spectrum of our cohort was comparable to previous CMML reports and dominated by frequent mutations in SRSF2 (46%), ASXL1 (45%) and TET2 (39%). Our analysis focused on genes analyzed in ≥90% of patients and found mutated in ≥10% (SRSF2, ASXL1, TET2, NRAS, RUNX1 and CBL). Mutation frequencies in these genes did not differ between AZA and DAC pts, nor between Sanger and NGS. In univariate analysis, a lower ORR was only seen in patients with ASXL1 mutations (ASXL1mut 44% vs ASXL1wt 62%, p=0.02) while only TET2 mutations predicted a higher CR rate (TET2mut 22% vs TET2wt 11%, p=0.02). Multivariate linear regressions confirmed that these differences were independent from clinical parameters. In univariate analysis, mutations in RUNX1 (13% of pts, HR=2.05, p=0.006) and to a lesser extent ASXL1 (45% of pts, HR=1.40, p=0.07) and CBL (12% of pts, HR=1.63, p=0.08) were associated with shorter OS. Similar results were found for AMLFS. There was no significant interaction between TET2 and ASXL1 or SRSF2 status in terms of ORR or OS. Clinical variables associated with poorer OS included splenomegaly (p=0.045), WBC > 13 x 109/L (p=0.003), platelets 2% (p=0.02), peripheral blasts > 1% (p=0.02) and high-risk karyotype (p=0.047). A multivariate Cox model retained RUNX1 mutations (p=0.007), splenomegaly (p=0.049), WBC > 13 x 109/L (p=0.005), platelets Conclusion: Contrary to previous results with smaller patient numbers (Braun et al Blood 2011), treatment with HMA seems to abrogate only partially the poor prognosis of ASXL1 mutations in CMML. RUNX1 mutations confer a poor prognosis in CMML treated with HMA and can be integrated with clinical and hematological parameters to improve prognostication of CMML pts treated with HMA, when compared with classical CMML risk scores. Disclosures Sallman: Celgene: Research Funding. Coombs: Pharmacyclics: Honoraria; H3 Biomedicine: Honoraria. Renneville: CELGENE: Research Funding. Platzbecker: Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding. Padron: Incyte: Honoraria, Research Funding. Santini: Celgene: Honoraria, Research Funding; Abbvie: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Amgen: Membership on an entity9s Board of Directors or advisory committees; Otsuka: Consultancy; Novartis: Honoraria. Fenaux: Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Itzykson: Janssen: Research Funding; Novartis: Research Funding.

Genomic Biomarkers to Predict Response to Hypomethylating Agents in Patients with Myelodysplastic Syndromes (MDS)

Genomic Biomarkers to Predict Response to Hypomethylating Agents in Patients with Myelodysplastic Syndromes (MDS)

Phase II Study of High Dose Cytarabine Followed By Pembrolizumab in Relapsed/Refractory Acute Myeloid Leukemia (AML)

Introduction: Outcomes are dismal for patients (pts) with relapsed/refractory (R/R) AML. Innate and adaptive immune aberrations exist in AML pts leading to immune escape. We have previously shown that there is an increased expression of multiple co-inhibitory molecules including programmed death-1 (PD-1) in the peripheral blood of AML pts at diagnosis, and these levels persist in pts with refractory disease (Knaus H, et al. Blood [Abstract] 2015). We hypothesized that administration of pembrolizumab after high dose cytarabine (HiDAC) salvage chemotherapy would stimulate a T-cell mediated anti-leukemic immune response leading to improved efficacy in R/R AML.Methods: A multicenter phase II study of age-adjusted HiDAC (<60 years: 2 gm/m2 IV Q12hours days 1-5; >=60 years: 1.5 gm/m2 IV Q12hours days 1-5) followed by pembrolizumab 200 mg IV on day 14 is being conducted in R/R AML pts 18-70 years. The primary endpoint of this study is the rate of complete remission (CR)/CR with incomplete recovery (CRi) after HiDAC followed by pembrolizumab. The study design is a Simon's like two-stage design (n=37 pts) with relaxed stopping for futility. Relaxed stopping refers to overall response rate (CR/CRi+partial remission [PR]) when stopping rule for futility is applied in the first stage. The null hypothesis that the CR/CRi rate for HiDAC followed by pembrolizumab is 20% will be tested against a one-sided alternative hypothesis yielding a type 1 error rate = 5% and power = 84% when the CR/CRi rate of HiDAC followed by pembrolizumab = 40%. Continuous monitoring for toxicity will be performed to assess for unacceptable toxicity. Overall responders will receive maintenance phase pembrolizumab 200 mg IV Q3weeks for up to 2 years until relapse/progression. Pts can proceed to allogeneic stem cell transplant (SCT) before or after maintenance phase (SCT must be >=30 days from last dose of pembrolizumab). Correlative studies include serial peripheral blood and bone marrow flow cytometry, mRNA-sequencing, and T cell receptor (TCR) clonality studies to determine predictive biomarkers of response.Results: 13 pts enrolled on this study between August, 2016-July, 2017 (Table 1). There have been 0 unacceptable toxicities to date. Immune-related adverse events have included grade 2 hyperbilirubinemia (n=1), grade 3 hepatic enzymes (n=1), both resolved promptly after systemic corticosteroids, as well as self-resolving grade 3 rash (n=1). Additionally, 1 pt developed grade 3 pulmonary edema without evidence of pneumonitis that resolved spontaneously without steroids. Of the 10 evaluable pts (as of July 1, 2017), overall response rate = 50% (CR/CRi: 4/10= 40%; PR = 1/10=10%), meeting the threshold to proceed to stage 2. Two pts have proceeded to a myeloablative allogeneic SCT in CR (one of whom received 2 cycles of maintenance pembrolizumab) and remain in CR. Post-SCT, both pts developed steroid-responsive acute GVHD of skin, one pt developed a transient increase in hepatic enzymes that was responsive to steroids, and one pt developed moderate chronic GVHD. Four pts have received maintenance pembrolizumab: 2 pts relapsed (median duration of CR = 3.9 months; range: 2-5.7 months), one pt proceeded to SCT after 2 cycles, and one pt initially achieved a PR and remains with stable disease after 11 cycles of maintenance pembrolizumab. No pts have died in remission. 30-day and 60-day mortality = 0% and 10%, respectively. Preliminary findings from the first 6 pts (3 responders vs. 3 non-responders) revealed increased diversity of TCR Vβ repertoire from pre-treatment peripheral blood CD8+ T-cells in responders when compared with non-responders using Shannon entropy analysis (p=0.0058; Figure 1).Conclusions: Early results of a multicenter phase II study demonstrate the feasibility of adding immune checkpoint blockade with pembrolizumab after HiDAC chemotherapy in R/R AML. An overall response rate of 50% is encouraging in a high-risk R/R AML cohort though ongoing findings are imperative to evaluate the efficacy and future role of pembrolizumab in AML. Interestingly, in a small cohort of patients, broadening of the immune repertoire, perhaps by increasing T cell responses beyond endogenous viral responses, was associated with response to PD-1 blockade. Future directions include a comprehensive immune biomarker discovery approach to determine predictors of response to immune checkpoint blockade in AML. [Display omitted] DisclosuresZeidner:Tolero Pharmaceuticals: Honoraria; Agios: Honoraria; Merck: Research Funding; Takeda: Research Funding; Tolero Pharmaceuticals: Research Funding; Celgene: Honoraria. Vincent:Merck: Research Funding; Pharmacyclics: Research Funding; Nanostring: Membership on an entity's Board of Directors or advisory committees. Foster:Shire: Honoraria; Incyte: Honoraria; Macrogenics: Research Funding; Celgene: Research Funding; Amgen: Honoraria; Pfizer: Research Funding; Celator: Research Funding. Coombs:Pharmacyclics: Honoraria; H3 Biomedicine: Honoraria. Gojo:Merck: Research Funding; Amgen: Research Funding; Amgen: Honoraria; Novartis: Research Funding. Serody:Merck: Research Funding.

Population-Scale Identification of Clonal Hematopoiesis Derived Mutations in Solid Tumor Mutation Profiling

▪Background: Clonal hematopoiesis (CH) is the commonly observed age-related acquisition of somatic mutations in hematopoietic lineage leading to clonal expansion of mutated cells. We recently showed 25% of advanced solid tumor patients harbor CH related mutations in their blood. As tumor tissue is typically infiltrated by blood cells, we hypothesized that sequencing tumors without the utilization of matched blood in a paired analysis could erroneously attribute CH mutations to be of tumor-specific origin. In review of patient clinical records, we encountered two patients (one endometrial adenocarcinoma and one mucosal melanoma) with DNMT3A R882H mutations reported by an external tumor-only NGS assay. In each case, paired tumor/blood sequencing subsequently confirmed the hematopoietic origin. Here, we show the prevalence of CH derived mutations in the solid tumors by analyzing a cohort of patients where tumor/normal sequencing is performed using MSK-IMPACT.Methods: We analyzed deep coverage, targeted next-generation sequencing data from January 2014 - May 2017 of paired blood and tumor samples from 15,432 patients with advanced solid malignancies using the MSK-IMPACT platform. MSK-IMPACT detects point mutations and indels in coding exons of currently 468 genes with older versions targeting 341 and 410 genes. We called mutations in blood DNA and identified those associated with CH using a custom filtering schema: First, germline mutations were eliminated based on variant allele fraction (VAF) > 0.35, except for mutations reported in COSMIC on at least 10 occasions. Second, we required mutations to be supported by at least 8 reads and present at twice the variant allele fraction (VAF) or greater in blood cells compared to the tumor. Third, we retained mutations that were detected with a VAF greater than 0.02 for a selected panel of leukemia genes and 0.05 for other genes. For cases where at least one mutation exceeded these thresholds, we reduced the VAF threshold in blood to 0.01 to detect subsequent events for leukemia genes and 0.03 for non-leukemia genes.Results: We identified 4,044 (26.2%) patients with at least one CH derived mutation in their blood corresponding to 6,588 presumptive somatic, non-silent mutations in 350 genes. Out of these, we identified 963 CH mutations from 808 patients (5.2%) that could have been classified as tumor-derived mutations in the absence of paired blood sequencing given a tumor VAF > 0.02. The number of mutations per patient ranged from 1-7 with a median VAF of 0.04 and 0.17 in paired tumor and blood samples, respectively. 34.5% of these variants are present in the COSMIC database with occurrence counts greater than 5 and include well-characterized driver alterations such as DNMT3A R882H, NRAS G12D, and IDH2 R140Q, which could readily be mistaken for somatic mutations in the corresponding solid tumor.Conclusion: With the potential for admixed leukocytes in biopsy specimens, these data demonstrate that failure to employ a paired blood sample in solid tumor sequencing can lead to the misclassification of CH mutations as somatic in the tumor, with clear diagnostic, prognostic, and therapeutic implications. DisclosuresCoombs:H3 Biomedicine: Honoraria; Pharmacyclics: Honoraria. Arcila:Raindance Tecnologies: Honoraria; Archer: Honoraria; Invivoscribe: Honoraria. Levine:Roche: Research Funding; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership.

Chimeric Antigen Receptor 4SCAR-GD2-Modified T Cells Targeting High-Risk and Recurrent Neuroblastoma: A Phase II Multi-Center Trial in China

Background: Neuroblastoma (NB) is one of the most common childhood malignancies responsible for 15% of pediatric cancer deaths. 70% of children with NB present with high-risk features characterized by severe metastasis and poor long-term survival even if the multimodal intensive combination therapies are used. There is ongoing challenge for developing novel therapeutic strategies for NB. GD2 is a well-characterized tumor antigen in NB. Anti-GD2 antibodies and T cells genetically modified to express GD2-specific chimeric antigen receptor (GD2-CART) have demonstrated anti-cancer activity. Here we report the safety and efficacy of a new generation of GD2-CAR (4SCAR-GD2) targeting high-risk NB with a 1-year follow-up (NCI registrations: NCT02992210 and NCT02765243).Patients and Methods: Total 34 high-risk NB patients, after confirmation for GD2 expression, are enrolled from 5 hospitals in China. These patients had undergone a series of standard therapies, including surgery, chemo- and radio-therapy before CART treatment. CD3 positive cells were selected from apheresis lymphocytes and activated before lentiviral infection. The cells were transduced with a caspase 9-inducible, safety-engineered lentivector CAR containing multiple intracellular signaling domains: GD2-scFv/CD28/CD27/CD3ζ-iCasp9. Before CART infusion, patients received cyclophosphamide and fludarabine conditioning. The CART infusion dose was at average 1.1x10^6/kg. Toxicity was determined based on NCI common toxicity criteria. The International Neuroblastoma Response Criteria were used to define response after CART treatment. The total observation time for all patients is 1 year after infusion.Results: GD2 expression was scored 0 to 4 by immunohistochemical staining of tumor sections, and total 142 specimens were stained. The results showed that 9% of specimens were negative and more than 71% scored more than 2. Total 34 patients were infused with 4SCAR-GD2 T cells: 24% of them experienced no adverse reactions (per NCI-CTC v.4.0), 50% had grade 1 and 26% had grade 2 toxicity responses including fever, skin rash and peripheral nerve pain. No grade 3 or 4 toxicity was observed. CART expansion kinetics were monitored by quantitative PCR of CAR in peripheral blood. The 4SCAR-GD2 illustrated long term persistence with the highest level of copy number detected at 30% on day 180 after CART infusion. Marked tumor regression was observed in 2 patients. At 28 days after infusion, by ultrasound the mediastinal and peritoneal tumors in one patient were reduced by 94% and 99.5%, respectively. The other patient had progressive tumor located in retroperitoneum and PET-CT showed shrinking tumor two months after CART treatment; retroperitoneal SUV decreased from 2.5 to 1.5. At the one-year observation point, 38% of patients were in stable disease, 15% had partial response and 47% were in disease progression. The 1-year overall survival rate is 74%.Conclusion: This multicenter trial demonstrates that the 4SCAR-GD2 T cells are safe and effective for treating high-risk stage IV NB children. The correlations of GD2 expression in tumor, CART kinetics in blood and treatment responses could not be statistically established, suggesting a vast disease heterogeneity. Important therapeutic correlates require a large patient cohort and long-term follow-ups to elucidate. DisclosuresYu:H3 Biomedicine Inc.: Employment.

Fatal COVID-19 is Characterized by Distinct Vasculopathic Features and Increased Congestion of the Alveolar Capillary-Bed

Background: Clinical and radiologic studies investigating Coronavirus disease 2019 (COVID-19) indicate that a possible cause of severe hypoxia is marked ventilation-perfusion (VA/Q) mismatch. Published histopathology reports diffuse alveolar damage (DAD) in fatal COVID-19 is indistinguishable from other causes of DAD. We compared lung parenchymal and vascular alterations between COVID-19 and DAD of other etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive COVID-19 patients (n=20) and patients with clinical acute respiratory distress syndrome and histologic DAD (n=21; non-COVID-19 viral and non-viral etiologies). Premortem chest CTs were evaluated for mosaic attenuation and vascular changes. Postmortem lung tissues were compared using histopathological, immunohistochemical, transcriptomics and computational analyses. Machine-learning-derived morphometric analysis of microvasculature was performed, with a random forest classifier quantifying vascular congestion (VSCong), in different microscopic compartments. Findings: On premortem CT, COVID-19 patients showed more mosaic attenuation (p<0·020), and subsegmental dilated vasculature (p=0·003) compared to controls. Histopathology revealed vasculopathic changes including: capillary-congestion and hemangiomatosis-like changes (p=0·043), microthrombi (p=0·026), thromboemboli (p=0·0036), infarcts (p=0·048), and perivascular inflammation(p <0·001). Immunohistochemistry revealed increased CD4+ T-cells(p=0·023), TIM3+ lymphocytes/macrophages (p=0·037/0·019), and PD-1+ cells(p=0·008). Morphometric analysis in COVID-19 revealed significantly higher bronchiolar VS Cong (p <0·0001) and alveolar-septal VSCong (p<0·0001), with larger overall VSCong range (p=0·002). Septal-congestion was associated with a significantly shorter time-to-death from symptom onset (p=0·02). Interpretation: Severe COVID-19 is characterized by significant vasculopathy and aberrant alveolar septal-congestion, which may be associated with shorter time-to-death. Dilated sublobar vessels and heterogeneously increased septal-congestion may translate to VA/Q mismatch in COVID-19. These findings warrant further investigation in larger multi-institutional autopsy cohorts. Funding Information: Supported by Internal Sundry, and Vickery-Colvin Award from the Department of Pathology, Massachusetts General Hospital. Declaration of Interests: LPH reports grants from Boehringer Ingelheim and has received personal consulting fees from Boehringer Ingelheim, Pliant Therapeutics, and Biogen Idec, all of which are not related to this study. SET have received CDC funding for other COVID-related work which are not related to this study. JAB has received grant support from Zeus Scientific, bioMerieux, Immunetics, the Bay Area Lyme Foundation and the Lyme Disease Biobank Foundation for work not related to this study, and has been a consultant for T2 Biosystems, DiaSorin and Roche Diagnostics, not related to this study. BPL reports royalties from Elsevier, Inc. for his work as an associate textbook editor, not related to this study. MMK has served as a compensated consultant for H3 Biomedicine and AstraZeneca and has received research (institutional) funding from Novartis and loyalty from Elsevier, all of which are not related to this study. The other authors declare no competing interests. Ethics Approval Statement: Analysis of patient autopsy material was reviewed and approved by the Partners Human Research IRB (Protocol #: 2020P001146).

A Phase 1 Study of Sea-CD70 in Myeloid Malignancies

Background SEA-CD70 is being developed in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Current treatment options are limited for patients (pts) with relapsed or refractory (r/r) MDS or r/r AML and outcomes remain poor. SEA-CD70 is an investigational humanized, non-fucosylated monoclonal antibody targeting CD70. Expression of CD70 is limited in normal tissue, but is aberrantly expressed on malignant myeloid blasts while absent from healthy hematopoietic progenitor cells. CD70 and its ligand, CD27, may play a role in malignant blast cell survival and/or tumor immune evasion. SEA-CD70 uses a novel sugar-engineered antibody (SEA) platform to produce a non-fucosylated antibody with enhanced effector function. The proposed mechanism of action of SEA-CD70 includes elimination of CD70 positive cells via enhanced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and mediation of complement-dependent cytoxicity (CDC). Additionally, SEA-CD70 has the potential to block the interaction of CD70 with CD27, which may disrupt signals that enhance blast proliferation and survival and may modulate the immune system to limit immune evasion and increase antigen specific T cell responses. Methods SGNS70-101 is a phase 1, open-label, multicenter, dose-escalation, and cohort expansion study designed to establish the safety, tolerability, and preliminary activity of SEA-CD70 in pts with myeloid malignancies (NCT04227847). Dose escalation is ongoing. In dose escalation, pts must have r/r MDS with 5-20% blasts which has failed prior treatment with a hypomethylating agent (HMA), and have no other therapeutic options known to provide clinical benefit for MDS. After conclusion of dose escalation, monotherapy expansion cohorts will be opened for pts with MDS and for pts with AML. Primary objectives are to evaluate the safety and tolerability, and to determine the maximum tolerated dose (MTD) or recommended expansion dose of SEA-CD70. Secondary objectives are to assess antitumor activity, PK, and immunogenicity of SEA-CD70. Once dose escalation is complete and the recommended monotherapy dose is identified, combination cohorts will be considered in AML and MDS. The study is currently enrolling with sites opening in the US and EU. Disclosures Aribi: Seattle Genetics: Consultancy. Advani:OBI: Research Funding; Takeda: Research Funding; Novartis: Consultancy, Other: advisory board; Pfizer: Honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Immunogen: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Donnellan:Kite Pharma/Gilead: Research Funding; Janssen: Research Funding; Karyopharm Therapeutics: Research Funding; AstraZeneca: Research Funding; Astex Pharmaceuticals: Research Funding; Incyte: Research Funding; MedImmune: Research Funding; TCR2 Therapeutics: Research Funding; Genentech: Research Funding; PTC Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Consultancy; Abbvie: Consultancy, Research Funding; Bellicum Pharmaceuticals: Research Funding; CTI Biopharma: Research Funding; Celgene: Research Funding; Celularity: Research Funding; Forma Therapeutics: Research Funding; Forty Seven: Research Funding; Takeda: Research Funding; H3 Biomedicine: Research Funding; Ryvu Therapeutics: Research Funding; Seattle Genetics: Consultancy, Research Funding. Fathi:Astellas: Consultancy; Agios: Consultancy, Research Funding; Amphivena: Consultancy, Honoraria; AbbVie: Consultancy; Pfizer: Consultancy; Daiichi Sankyo: Consultancy; Celgene: Consultancy, Research Funding; Forty Seven: Consultancy; Jazz: Consultancy, Honoraria; Kite: Consultancy, Honoraria; NewLink Genetics: Consultancy, Honoraria; Novartis: Consultancy; PTC Therapeutics: Consultancy; Takeda: Consultancy; TrovaGene: Consultancy; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Blue Print Oncology: Consultancy; Boston Biomedical: Consultancy; Kura: Consultancy; Trillium: Consultancy; Seattle Genetics: Consultancy, Research Funding. Rotta:Merck: Speakers Bureau; Jazz Pharma: Speakers Bureau. Vachani:Blueprint: Consultancy; CTI Biopharma: Consultancy; Daiichi Sankyo: Consultancy; Incyte: Consultancy, Research Funding; Jazz: Consultancy; Astellas: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy; Abbvie: Consultancy. Yang:AROG: Research Funding; Protagonist: Research Funding; Jannsen: Research Funding; AstraZeneca: Research Funding. Ho:Seattle Genetics: Current Employment, Current equity holder in publicly-traded company. Garcia-Manero:Novartis: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Merck: Research Funding; Jazz Pharmaceuticals: Consultancy; Onconova: Research Funding; Amphivena Therapeutics: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; AbbVie: Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; H3 Biomedicine: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Genomic and Transcriptomic Differences of Myelodysplastic Syndrome/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis (MDS/MPN-RS-T) and Myelodysplastic Syndrome with Ring Sideroblasts (MDS-RS)

INTRODUCTION: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a rare hematological disorder characterized by anemia, bone marrow dysplasia with ring sideroblasts and persistent thrombocytosis, and high frequency of SF3B1 and JAK2 mutations. Despite clinical, histological and molecular similarities with MDS with ring sideroblasts (MDS-RS), the clinical outcomes of these entities are diverse. To date, there is no data evaluating specific functional pathways which might explain phenotypic and clinical differences beyond diverse frequencies of JAK2 mutation. METHODS: We evaluated a total of 24 patients (pts) with MDS/MPN-RS-T and 27 pts with MDS-RS. Diagnosis was based on WHO 2017 criteria and confirmed by two independent hematopathologists. Whole bone marrow DNA was subject to 81 gene targeted next-generation sequencing (NGS) analysis. CD34+ cells from bone marrow samples of 4 pts with MDS/MPN-RS-T, 7 pts with MDS-RS and 17 healthy individuals obtained from AllCells (Emeryville, CA) were isolated using the CD34 MicroBead Kit and RNA was isolated using the PicoPure RNA isolation kit. Fastq files were mapped to the human genome (build GRCh38) in TopHat2 using the default options. Differential gene expression analysis was conducted using DESeq2 in R version 3.6.2. Pathway enrichment analysis was performed using gene set enrichment analysis, with the fgsea library in R. RESULTS: Patients with MDS/MPN-RS-T had higher median bone marrow ring sideroblast percentage (47% vs 32%, p=0.04) and absolute neutrophil count (4.34x109/L vs 2.99x109/L, p=0.001). Frequency of identified mutations and their VAFs compared to MDS-RS are shown in Figure 1A. The median number of mutations was higher in MDS/MPN-RS-T than in MDS-RS (3 vs 2, p&amp;lt;0.001). SF3B1 mutations were the most frequent in both entities (MDS/MPN-RS-T: 92%, MDS-RS: 82%), had similar median VAF (34% vs 32%, p=0.619), and involved the hot spot codon K700E in 64% and 43% of MDS-RS and MDS/MPN-RS-T (p=0.227), respectively. As expected, 58% of pts with MDS/MPN-RS-T had JAK2 V617F mutations but were also more likely to have mutations in kinase signaling genes (NF1, SETBP1, CBL, CBLB, FLT3 TKD, MPL) compared to MDS-RS (29% vs 4%, p=0.019). Four (40%) of JAK2 negative MDS/MPN-RS-T had mutations in kinase signaling genes. There were no differences in frequency of TET2 mutations between both entities. However, there was a trend for the median VAF of TET2 mutations in MDS/MPN-RS-T to be lower than in MDS-RS (1.5% vs 21.1%, p=0.177) suggesting a likely subclonal nature of these mutations compared to MDS-RS in which they appeared as dominant events. MDS/MPN-RS-T showed distinct transcriptomic profile compared to both healthy controls and MDS-RS. Compared to healthy controls, a total of 2 pathways were significantly upregulated and 58 were downregulated in MDS/MPN-RS-T while 5 pathways were upregulated and 69 were downregulated in MDS-RS. Compared to MDS-RS, a total of 29 pathways were significantly upregulated and 26 were downregulated in MDS/MPN-RS-T. The most significantly upregulated pathways in MDS/MPN-RS-T included genes involved in platelet activation and aggregation, cytokine signaling, and signaling through GPC receptors (Figure 1C). Compared to both healthy control and MDS-RS, MDS/MPN-RS-T was characterized by downregulation of genes involved in DNA damage response, regulation of apoptosis, telomere maintenance and RNA synthesis (Figure 1D). MDS-RS was characterized by downregulation of genes involved in signaling by GPC receptors and MAPK signaling, mRNA splicing, cytokine signaling and signaling through interleukins compared to both control and MDS/MPN-RS-T (Figure 1C). CONCLUSIONS: MDS/MPN-RS-T is characterized by co-dominance of SF3B1 and JAK2 mutations and presence of minor kinase signaling mutations not observed in MDS-RS. Upregulation of cytokine and interleukin signaling mediated through GPC receptors, and downregulation of genes involved in apoptosis and DNA damage are unique transcriptomic features of MDS/MPN-RS-T likely driven by genotype. These unique genomic and transcriptomic characteristics of MDS/MPN-RS-T supports the classification of MDS/MPN-RS-T based on genomic features beyond presence of SF3B1 mutation, and might represent potential therapeutic avenues for this rare disease. Figure Disclosures Sasaki: Otsuka: Honoraria; Pfizer Japan: Consultancy; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy. Kantarjian:Sanofi: Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria; BMS: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotechnologies: Honoraria; Aptitute Health: Honoraria; Immunogen: Research Funding; Jazz: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BioAscend: Honoraria; Novartis: Honoraria, Research Funding; Delta Fly: Honoraria; Pfizer: Honoraria, Research Funding; Oxford Biomedical: Honoraria; Ascentage: Research Funding. Garcia-Manero:Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding; Onconova: Research Funding; Merck: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy.