Leukemia Samples Research Articles

Immunopeptidomics-Guided Warehouse Design for Peptide-Based Immunotherapy in Chronic Lymphocytic Leukemia

Antigen-specific immunotherapies, in particular peptide vaccines, depend on the recognition of naturally presented antigens derived from mutated and unmutated gene products on human leukocyte antigens, and represent a promising low-side-effect concept for cancer treatment. So far, the broad application of peptide vaccines in cancer patients is hampered by challenges of time- and cost-intensive personalized vaccine design, and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. In this study, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics. Comparative mass spectrometry-based immunopeptidome analyses of primary chronic lymphocytic leukemia (CLL) samples, as representative example of low-mutational burden tumor entities, and a dataset of benign tissue samples enabled the identification of high-frequent non-mutated CLL-associated antigens. These antigens were further shown to be recognized by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, and were evaluated as pre-manufactured warehouse for the construction of personalized multi-peptide vaccines in a first clinical trial for CLL (NCT04688385). This workflow for the design of peptide warehouses is easily transferable to other tumor entities and can provide the foundation for the development of broad personalized T cell-based immunotherapy approaches.

Abstract 2259: Single-cell multi-omics analysis of SNV, CNV, and protein expression

Abstract Recent advancements in precision medicine, while highly promising, presents a major technical challenge to researchers due to disease heterogeneity. The emergence of single-cell technologies has greatly refined the resolution in which sample diversity can be investigated, enhancing the efficiency of selecting appropriate molecular targets. Additionally, applying multi-omics analysis on single cells would further improve the understanding of cell-to-cell heterogeneity by providing unique insights on cellular and genetic composition. Using a two-step droplet microfluidic technology, the Mission Bio Tapestri Platform enables multiplex-PCR based high-throughput targeted DNA sequencing in single cells to obtain single-nucleotide variation (SNV) and copy number variation (CNV) information. By leveraging this technology, a new workflow is developed to detect protein expression in addition to DNA genotype in the same single cells. In this approach, cells are labeled with a pool of oligonucleotide-conjugated antibodies prior to loading the cells into the Tapestri Instrument. Sequencing libraries are then prepared from both antibody oligonucleotides and the amplified DNA sequences, followed by identification of single-cell DNA genotypes and protein signatures from the sequencing readout. The number of protein targets can be in the range from 6 to over 40, which is beyond the limit for a single flow cytometry run. This method has been successfully performed on cell lines, fresh and frozen PBMCs, as well as clinical samples. In an acute myeloid leukemia (AML) sample, combined single-cell SNV, CNV, and protein expression data illustrated the heterogeneity within the sample. The data clearly identified CD3+ T cells and CD19+ B cells without pathogenic SNVs and CNVs. CD34hiCD11blo and CD34loCD11bhi subpopulations were also identified within the cells carrying the same pathogenic SNVs and CNVs. We believe that this novel multi-omics technology will facilitate new discoveries in the complex relationship between genotype and phenotype, enable a better understanding of disease biology, and subsequently improve the design of diagnostics and therapies. Citation Format: Aik Ooi, Dalia Dhingra, Adam Sciambi, Kate Thompson, Jacqueline Marin, Saurabh Parikh, Mani Manivannan, David Ruff. Single-cell multi-omics analysis of SNV, CNV, and protein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2259.

Simultaneous Analysis of Telomere Lengthes and Cell Surface Antigen in Leukemia by Multicolor Flow-FISH

During the last decade telomere length (TL) and telomerase have been discovered as new biological markers and potential new targets of cancer including malignant hematopoietic disorders. A FLOW-FISH method, i.e. quantitative fluorescence in situ hybridization (Q-FISH) in combination with flow cytometry (FCM), has been developed recently. Most overseas studies detect TL by FLOW-FISH without combined analysis of cell immunophenotype. We analyzed TL and cell immunophenotype simultaneously in the same sample by FLOW-FISH in order to combine the two markers to found one generally applied method for monitoring disease conditions or predicting clinical outcome in leukemia.Methods:HL60, Raji, Molt4 cells were cultivated. Each step and the conditions of the FLOW-FISH procedure were optimized, standardized and validated. The relative TL (RTL) of the leukemic sample cells compared to the control cells(Molt4).BM samples were derived from 34 leukemia patients. 20 normal PB samples from healthy individuals were set as control group.The BM or PB MNC were collected and used for FLOW-FISH. Meanwhile, the RQ-PCR measurements in the same samples were performed to test the correlation of the two methods.Results:Using various cell lines, multicolor FLOW-FISH was also successfully established.The median of RTL in 24 AL patients was 0.240, 2 CLL was 0.235, 3 CML-BP was 0.249, 5 CML-CP was 0.435, while that in 20 normal controls was 0.408.(P=0.001). The TL was shorter in patients with AL, CLL, CML-BP than in normal controls, otherwise, the CML-CP cases had no difference with the controls. 14 AL patients had the significantly longer telomere with clinical remissions than at impetus stage (P=0.01), and no significant difference was found between the 12 cases with clinical remissions and the normal controls(P=0.363). A high degree of correlation between the RTL obtained from FLOW-FISH and TL estimation by RQ-PCR existed (P=0.000).There was a significant negative correlation between TL and age in 20 normal controls(P=0.001), and the female had longer telomere than the male(P=0.037). However no statistic difference between TL and age or gender existed in 34 de novo leukemia patients. The TL of 24 AL cases had a negative correlation with peripheral white cells counts(P=0.002). Within 23 follow-up AL patients, the CR rate in cases with TL above the median was 81.82%, and 58.23% in the rest. Among 5 CML-CP patients who received imatinib treatment, 2 cases with the shorter telomere occurred transformation to BP in less than 6 months, the other 3 cases obtained CHR within 3 months, CCyR at 6 months and no transformation to BP was observed in our follow-up of 10 months.14 AL patients who undergone simultaneous analysis of TL and immunophenotype show the coincidence with the convention immunology in cell differentiation antigen expression. TL combined with cell differentiation antigen of 5 cases were dynamically detected at different courses of disease. It showed that TL of 2 cases with continued CR restored after CR and remained at normal level during remission, and no specific antigen expressed highly. However, telomere of 3 relapsed cases were significantly shortened at impetus stage, prolonged after CR and shortened again after relapse. In 2 relapsed cases, the telomere of related antigen positive cells shortened before the whole cells, and ahead of cell morphology change.Conclusions:The TL was significantly shorter in 34 de novo leukemia patients than in normal controls except the CML-CP cases. Restored telomeres were found with clinical remissions in 14 AL cases. The two methods of FLOW-FISH and RQ-PCR generated the same results and showed notable correlated.There was a significant negative correlation between TL and age in normal controls, and the female had longer telomere than the male. However no statistic difference between TL and age or gender existed in de novo leukemia patients. The TL of 24 AL cases had a negative correlation with peripheral white cells counts. It seemed the shorter telomere mean the lower CR rates in 23 follow-up cases with AL and the shorter duration of CP before onset of BP in 5 cases with CML.Simultaneous analysis of TL and cell differentiation antigen by multicolor FLOW-FISH made for overall monitor for leukemia and might be a new generally applied method for monitoring MRD in leukemia.

Histone Modification Patterns Using RPPA-Based Profiling Predict Outcome in Acute Myeloid Leukemia Patients

Background: Prognostic factors based on cytogenetic and molecular abnormalities combined with clinical features like age, performance status, or history of an antecedent hematological disorder, are currently used to predict clinical outcome in acute myeloid leukemia (AML). (Döhner H. et al. 2010) Although, sequencing of AML has identified many driver mutations in genes associated with epigenetic regulation, e.g. post-translational histone tail modifications. Altered activity of post-translational histone tail modifications has been shown to contribute to AML leukemogenesis via regulation of gene transcription. (Fong C.Y. et al. 2014) The functions and effects of modulating histone-modifying proteins (HMP) are already typically studied by altering an individual protein of interest. However in AML, it is the net consequence of the combined influences of all the HMP, rather than a single protein, that determines the net effect on the cell. A global analysis of the level and patterns of expression of multiple HMP and the effect of different patterns of expression on outcome and prognosis has not been investigated in AML patients.Methods: We analyzed 20 HMP by reverse phase protein array (RPPA) in a cohort of 205 newly diagnosed AML patients evaluated at the University of Texas M.D. Anderson Cancer Center. (Kornblau S.M. et al. 2009) Protein levels were correlated with patient and disease characteristics, including survival and mutational state.Results: Different protein clusters were identified and characterized by higher (more on) or lower (more off) expression of HMP, relative to normal CD34+ cells (figure 1A). We found that the more on state of HMP was associated with poorer overall survival (P= 0.007, figure 1B) and more chemo resistance (P < 0.001) compared to normal -like and a more off state. Furthermore, FLT3 mutated AML patients were significantly overrepresented in the more on state (P= 0.025). Identification of the markedly upregulated proteins: KDM1A, hnRNPK, NCL, SIRT1, ASH2L, WTAP in all, and BRD4 and NPM1 in some on state patients suggest targets to develop drugs against that may be the most efficacious when used individually or in combination.Conclusions: These findings showed for the first time that multiple HMP form recurrent patterns of expression and that these significantly correlate with prognosis, chemo resistance, and FLT3 mutational state in newly diagnosed AML patients.F igure legend 1:(A) RPPA-based heatmap containing 20 histone modification related proteins showing only 205 fresh AML patient samples. Clusters were determined by a progeny clustering based algorithm coupled with k-means, which computationally calculated the optimal number of five clusters. The five cluster colors along the top bar delineate the five fresh clusters. Cluster 1 (red) was defined as a normal -like pattern by principal component analysis, whereas clusters 2, 3, 6 and 7 were only seen with leukemic samples. Expression is normalized for each protein to range from the lowest (blue) to the highest (red). Cluster 2 and 7 were defined as the more on clusters and cluster 3 and 6 as the more off clusters.(B) Overall Survival of patients from the normal -like histone-modifying proteins (HMP) cluster (n=61), more on HMP clusters (n=131) and the more off HMP clusters (n=13) as determined in our cohort of newly diagnosed AML fresh patient samples by RPPA-based profiling. The survival of patients with the more on state of HMP was significantly reduced compared to the more off state and the normal- like HMP signatures. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genome-Wide Analysis of the Regulatory Landscape in Different Pre-B-ALL Subtypes

BackgroundAcute pre-B leukemia results from a failure in cell differentiation. In normal development, cells commit to their lineage and differentiated phenotype state by the activation of transcription factors (TFs), or TF modules, in a series of decision points at which a choice is made between alternative lineage fates. Previously, we showed that the ETV6-RUNX1 gene translocation that occurs in 25% of pediatric leukemia patients silences regulatory regions containing RUNX binding sites (enhancers) at target gene loci implicated in lymphoid lineage differentiation (Teppo et al. 2016). However, the genome-wide changes in enhancer activities at binding sites of other TFs functioning in B-cell differentiation remain poorly understood. Moreover, the genome-wide identification of enhancers altered during leukemogenesis is incomplete.Material & MethodsTo measure transcripts generated both at enhancer and gene regions, global run-on sequencing (GRO-seq) technique was used to measure nascent RNAs (Heinäniemi et al. 2016) from human pre-B-ALL patient samples (N=35) and several cell lines representing different subtypes. For validation, additional samples were generated from cell line models with overexpression or silencing of leukemic TFs. To this collection of 151 nascent transcriptomes, additional 92 GRO-seq samples representing different cell lineages (Liivrand et al 2017) were used to compare the leukemic cells to primary cells and other cancers. Enhancer RNA (eRNA) levels were compared at TF binding motif centered enhancer regions. TF ChIP-seq data from leukemic cells and HSC were acquired from the NCBI Gene Expression Omnibus for validation of this approach.ResultsTo gain insight into the derailed regulatory landscape in leukemic cells, we identified eRNA regions both from normal and leukemic samples and performed statistical analysis distinguishing regulatory regions with differential activity between pre-B-ALL-subtypes or compared to normal CD34+ bone marrow precursors. Next, association of the significantly altered enhancers with gene transcripts was utilized to build regulatory maps for differentially expressed gene loci. For this purpose, we developed a transcript identification and quantification pipeline suitable for detecting altered expression at different coding and non-coding RNA loci from nascent transcriptomes (Liivrand et al 2017; unpublished). To globally evaluate altered TF activities across pre-B-ALL subtypes underlying the differences observed, we present here an unbiased approach based on eRNA quantification at TF motif-centered enhancers. The analysis of eRNA levels at binding sites of TFs with established role in leukemia was used as a benchmark: we could confirm low eRNA transcription at RUNX1 binding sites in the ETV6-RUNX1 subtype, elevated eRNA levels at HOXA9 and MEIS1 binding sites in the MLL rearranged subtype, while TCF3, BCL6 and PBX1 bound sites were most active in TCF3-PBX1 samples.ConclusionsOur results provide the first genome-wide transcriptional regulatory landscape of different pre-B-ALL subtypes. We identified novel subtype-specific active enhancers and annotated regulatory non-coding regions that can be utilized to evaluate the functional impact of genetic alterations outside gene regions. DisclosuresNo relevant conflicts of interest to declare.

Progression to AML Is Predictable and Distinct from Age Related Clonal Hematopoiesis

Introduction: Despite marked improvements in our understanding of the biology and genetic basis of acute myeloid leukemia (AML) gained in the past 4 decades, the overall survival of AML has only improved slightly. The strategy for targeting AML once the disease is diagnosed remains grim due to subclonal genetic diversity and the stemness properties of relapse originating cell types. As studies in solid tumors have shown, strategies focused on targeting the cancer at earlier stages hold considerable promise, however no predictive tests are available for AML. The existence of clonally expanded pre-leukemic hematopoietic stem and progenitor cells (preL-HSPC) within the diagnostic blood sample of AML patients provides strong evidence that the disease must evolve over long time periods prior to diagnosis. However, with the discovery that age related clonal hematopoiesis (ARCH) is exceedingly common during normal aging; a refined search for the factors that distinguish ARCH from those rare individuals who will actually develop AML had to be taken.Methods: We hypothesized that the basis for clonal expansion in ARCH was different than in the clonal expansion leading to AML. To investigate genetic differences, we developed sensitive and accurate error correction sequencing method to study blood samples of healthy individuals from the EPIC cohort before AML diagnosis. These 96 pre-AML cases donated their blood on average 6 years before eventually being diagnosed with AML; a set of 420 matched controls were sequenced in the same way. Furthermore, 3.2 million electronic health records (EHR) from the Clalit database were inspected to identify non-genetic AML-risk prediction parameters.Results: The overall variant allele frequency (VAF) distribution of the identified mutations was significantly different between cases and controls. Specifically, a greater proportion of cases carried somatic mutations with intermediate-high VAF (70.8% versus 36.6% in the controls). The median number of all somatic mutations was significantly higher in the cases as compared with the controls (7 and 5 respectively) and was correlated with aging in both groups. Interestingly, the pre-AML group had a higher rate of mutation accumulation compared to controls, resulting in significantly greater numbers of mutations occurring after the age of 55. Recurrent AML mutations were more prevalent in cases compared to controls (Age>=55: OR, 7.571; 95% CI, 4.055-14.387). Specifically, highly recurrent splicing factors mutations (e.g. SRSF2 P95H, U2AF1 Q84P, and SF3B1 K700E) were found solely in the pre-AML group suggesting that specific mutations that rewire the splicing machinery inevitably leads to AML when acquired in HSPC. The VAF of recurrent AML mutations was significantly higher in the cases compared to controls. Furthermore we observed a higher proportion of individuals with more than one recurrent AML mutation in the pre-AML group compared to controls (OR, 14.316; 95% CI, 6.318-34.933). Incorporating these factors, we construct a computational model for predicting progression toward AML. The AML risk prediction model predicted progression on average 7 years before actual diagnosis (HR, 20.1, 95% CI 8.59-46.8). EHR data was found to be highly valuable for the identification of high-risk population. Compared to controls, AML patients (N=982) showed aberrant blood counts up to 6 months prior diagnosis (relative risk, 22.2).Conclusions: These results reveal that the clonal expansion occurring during normal aging is highly distinct from pre-AML clonal expansion, which has a highly predictable evolutionary trajectory. Our study provides clear proof of concept for the feasibility of early AML prediction based on somatic mutation patterns, yet, since ARCH is common, the real-world feasibility of applying our AML risk prediction model will require identifying higher-risk individuals through routine clinical testing. We have shown that information obtained from EHR is useful for that purpose. Future studies of independent population cohorts with access to serial viable blood and leukemia samples will allow for incorporation of information such as the specific identity of the mutated cells and clone expansion kinetics. Such studies will aid in developing better understanding of which individuals should be selected for future clinical trials to study the potential benefits of early interventions in this deadly disease. DisclosuresNo relevant conflicts of interest to declare.

The Role of Kdm6a in Malignant Hematopoiesis

Our preliminary studies identified recurrent, somatic mutations in Kdm6a in a mouse model of acute promyelocytic leukemia (APL). Putative inactivating mutations of Kdm6a (primarily small deletions) occurred in approximately 50% of mouse APL samples, making these the most common acquired progression events in this model. We found somatic mutations of KDM6A in 4 of 200 acute myeloid leukemia (AML) samples. However, in a cohort of 42 patients with APL, we identified no somatic alterations in KDM6A . To better understand the role of Kdm6a in malignant hematopoiesis, we intercrossed our Kdm6a conditional knockout (KO) x Vav1 - Cre mice with a “knock-in” mouse model of APL (Ctsg-PML-RARA mice). To determine whether Kdm6a inactivation cooperates with PML - RARA, we performed a tumor watch that included all genotypes of Kdm6a conditional KO female and male triple cross mice. After 18 months of observation, we found that the latency of APL was significantly decreased in all three cohorts of Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (Figure 1). This finding was most pronounced in female homozygous Kdm6a KO triple cross mice, which had a median survival of 161 days versus 365 days in the control cohort (P < 0.01 by log-rank test). The Kdm6a -null mice developed the typical phenotype of mouse APL, including splenomegaly, myeloproliferation and leukemic cells that coexpressed Gr-1 and Kit. The triple cross APL cells remained sensitive to all- trans retinoic acid (ATRA) and arsenic trioxide ex vivo . Compared with 56% penetrance in Ctsg-PML-RARA mice, female homozygous and male hemizygous Kdm6a KO x Vav1 - Cre x Ctsg-PML-RARA mice developed APL with 100% penetrance. We fully characterized pre-leukemic hematopoiesis in young (6 to 8-weeks-old) Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (compared to appropriate littermate controls) to define the underlying mechanism(s) by which Kdm6a inactivation contributes to leukemogenesis. Like our double cross Kdm6a conditional KO x Vav1 - Cre mice, the loss of Kdm6a expression in young female homozygous triple cross mice caused mild thrombocytopenia and splenomegaly. These young mice had neutrophilia and lymphopenia, which the female Kdm6a -null double cross mice only acquired with age. We observed a trend for increased numbers of colonies in a myeloid serial replating assay that was also specific to this cohort. Next, we demonstrated that this cohort had significant decreases in the Kit+Lin-Sca-1+ (KLS) cell, short-term hematopoietic stem cell (ST-HSC), and multipotent progenitor (MPP) hematopoietic stem and precursor compartments. This recapitulated the findings from the young, double cross Kdm6a -null female mice but was more pronounced. We found a concomitant increase in granulocyte-macrophage progenitors (GMPs) and decrease in megakaryocyte-erythroid progenitors (MEPs) that was also specific for the female Kdm6a -null triple cross cohort. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged (1-year-old) female Kdm6a -null double cross mice and aged controls. Unsupervised hierarchical clustering revealed a clear distinction between cohorts. We identified 1520 differentially expressed genes by an ANOVA analysis (fold change > 2, P < 0.05) between the Kdm6a -null versus WT aged SLAM cells. The list of differentially expressed genes included Pml (downregulated in Kdm6a -null SLAM cells with fold change of 2.35) and Flt3 (upregulated in Kdm6a -null SLAM cells with fold change of 2.32). Expression changes of these two genes may be associated with the mechanism of how inactivation of Kdm6a cooperates with PML-RARA in our mouse model. Pml haploinsuffiency has been show to modestly cooperate with PML-RARA in mouse models of APL, and FLT3 mutations are the most common secondary mutations in APL. In an effort to further elucidate the mechanism by which loss of Kdm6a contributes to malignant hematopoiesis, we have completed RNA-Seq, ATAC-Seq, anti-histone H3 (monomethyl K4) ChIP-Seq, and anti-histone H3 (trimethyl K27) ChIP-Seq studies on mouse APL samples derived from Ctsg-PML-RARA mice that developed spontaneous leukemias (n = 3 Kdm6a -WT samples versus n = 3 Kdm6a -mutant samples). Analysis of this data set is now underway. In sum, our data have demonstrated a functional role of Kdm6a in malignant hematopoiesis and have improved our understanding of its subsequent connection to leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Stromal Cell Mediated Resistance to Mertk Inhibition in Acute Myeloid Leukemia

MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by small molecule tyrosine kinase inhibitors (TKIs), UNC2025 or MRX-2843, altered cell cycle progression and induced apoptosis in leukemia cell cultures. Moreover, treatment with these MERTK TKIs prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with the TKI effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance.To evaluate the role of the bone marrow stromal niche in mediating resistance to MERTK inhibition by MRX-2843, AML cell lines were cultured in the presence of a fibroblast-like cell line (HS27) and induction of cell death and apoptosis was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 200nM MRX-2843 compared to leukemia cells alone (31.9% versus 61.2%, p<0.05). Similar results were observed in NOMO-1 and OCI-AML5 AML cell cultures. Additionally, cell cycle profiles were determined by flow cytometry in the presence or absence of stromal cells and MRX-2843. Treatment of Kasumi-1 cell cultures with 200nM MRX-2843 in the absence of stromal cells resulted in a significant accumulation of cells in G2 phase relative to vehicle-treated cultures (8.9% versus 14.8%, p<0.01) and was accompanied by an increase in γH2AX expression detected by immunoblot. In contrast, there was no change in cell cycle progression or γH2AX expression in cultures treated with MRX-2843 when co-cultured with the stromal cell line. NOMO-1 cells also accumulated in G2 phase in response to treatment with MRX-2843 and were protected from changes in cell cycle progression by co-culture with HS27 cells. Additionally, MERTK inhibition resulted in induction of polyploidy in NOMO-1 cell cultures, but this effect was abrogated in the presence of stromal cells. Collectively these results indicate abrogation of cell cycle progression in leukemia cells in response to treatment with MRX-2843 and demonstrate protection from this effect mediated by stromal cell interactions.The MERTK ligand, GAS6, mediates resistance to cytotoxic chemotherapy in leukemia cells and is a poor prognostic marker in AML. To evaluate the role of the MERTK ligands GAS6 and PROS1 in resistance to MERTK inhibition, their expression in stromal cells was determined by immunoblot. GAS6, but not PROS1, was expressed in HS27 cells; however, MRX-2843 retained its ability to inhibit MERTK phosphorylation in stromal cell co-cultures. We hypothesized that upregulation of the other TAM kinases, AXL and/or TYRO3, may mediate resistance as they share common ligands. To test this hypothesis, Kasumi-1 cells were treated with UNC4241, a small molecule pan-TAM inhibitor, in the presence of stromal cells. While co-culture protected Kasumi-1 cells from MRX-2843 induced apoptosis, treatment with UNC4241 induced apoptosis in the presence of stromal cells (32.7% versus 20.4% in vehicle control, p<0.01).To further evaluate the interactions between stromal cells and leukemia cells, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (74.0% vs. 69.8% in unconditioned medium, p>0.05). Similarly, addition of 500nM exogenous GAS6 or PROS1 did not protect leukemia cells from apoptosis in the presence of MRX-2843. Together, these results suggest that direct cell-cell contact between leukemia and stromal cells is necessary to provide protection.Together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from MRX-2843 induced apoptosis and cell cycle alterations and suggest that bone marrow mobilizing agents may be effective to overcome resistance to MERTK inhibitors. DisclosuresWang:Meryx: Equity Ownership; University of North Carolina: Patents & Royalties. Frye:University of North Carolina: Patents & Royalties; Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Earp:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. DeRyckere:Meryx: Equity Ownership. Graham:Meryx: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Impact of IRF4/MUM1 Expression on Adult T-Cell Leukemia/Lymphoma and Treatment Outcome

Introduction:HTLV-1 related adult T-cell leukemia-lymphoma (ATLL) remains fatal, thus urging development of new therapies. Interferon regulatory factor 4 (IRF4), also known as MUM1, is a transcription factor expressed in various aggressive B-cell and T-cell lymphomas (1). IRF4 has been reported to be highly expressed in HTLV-1 transformed cells and in aggressive leukemic forms of ATLL, and has also been implicated in interferon resistance. However, IFR-4/MUM1 expression, and its pathogenic role in ATLL have not been fully investigated. We previously reported lack of IRF4/MUM1 in leukemic ATLL specimens from patients who responded to AZT-interferon-alpha (AI). In this study, we characterized the expression of IRF4/MUM1 in our expanded ATLL cohort.Methods:ATLL specimens from 73 chemotherapy naïve patients of all subtypes [lymphomatous (L) n=31, acute (A) n=36, unfavorable chronic (UC) n=5, and smoldering (S) n=1] were analyzed for MUM1 expression. Cases were analyzed or confirmed by immunohistochemistry at our institution using anti-human MUM1 antibody (Dako) on formalin-fixed paraffin-embedded tissue or cellblock sections prepared from cytospun CD4+-enriched leukemic samples. The cutoff positive value for MUM1 expression was 30%. MUM1 expression was correlated with treatment outcome.Results:In 73 patients the proportion of MUM1+ cases was as follows: L (28/31 =90%), A (7/36 =19%), UC (1/5 =20%), and S (0/1 =0%) (Figure 1A). MUM1 expression was analyzed in 33 chemotherapy naïve patients with A (n= 28) or UC type (n=5) treated with AI; only 3 patients (A n=2, and UC n=1) were MUM1+ and had partial response (PR) (UC n=1), stable disease (SD) (A n=1), and no response (NR) (A n=1) respectively (Figure 1B). All 9 patients who achieved a complete response (CR) (A n=7, and UC n=2) and 6 patients with PR (A n=5, UC n=1) were MUM1-. The cumulative 3-year overall survival (OS) rates in MUM1- (n=37) vs. MUM1+ (n=36) patients were 23% vs. 10% respectively (p=0.68) (Figure 2). Notably, the great majority of patients in the MUM1- group (33/37=89%) were treated with AI as first line therapy and were A-type (27/37=73%), while most patients in the MUM1+ group (26/36=70%) were treated with chemotherapy upfront and were L-type (23/36=64%). The cumulative 3-yr progression-free survival in MUM1- patients treated with AI was 28% as compared to 0% in MUM-1+ treated with chemotherapy (Figure 3).Conclusion:Our results demonstrate that MUM1 expression is highly associated with L-type ATLL. Patients with aggressive leukemic ATLL subtypes treated with AI generally lacked MUM1 and had a significant better outcome than L-type patients treated with chemotherapy. This study suggests that MUM1 expression may influence the biology at ATLL subtypes and response to interferon-based therapy. Targeting IFR-4/MUM1 function in L-type ATLL should be considered as a therapeutic strategy. The analysis of additional cases is still ongoing and the final results will be presented at the meeting. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PDX Models Recapitulate Genetics and Epigenetics of Pediatric T-Cell Leukemia

Patient-derived xenografts (PDX) emerged as important tools to study leukemia biology and potential therapeutic regimens. Here, we compared cellular, molecular and developmental features of PDX-derived blasts with primary pediatric precursor T-cell acute leukemia (T-ALL) cells. The cells were obtained at the times of initial diagnosis and relapse. We analyzed the samples at the genomic level (whole genome sequencing (WGS), whole exome sequencing (WES), multiplex ligation probe amplification (MLPA), targeted sequencing) and the epigenetic level (DNA methylation, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq)).Altogether 75% of our samples engrafted successfully. Of these 12 were matched pairs of initial diagnosis and relapse as well as corresponding PDX. Of further 12 matched pairs either initial diagnosis (2/12) or relapse (7/12) or neither (3/12) engrafted indicating that relapse samples tend to engraft more successfully (p= 0.1824 (McNemar's test)), which is in agreement with observations in other cancer entities.In 11 out of 12 matched pairs (initial and relapse) of primary and PDX genomic stability of the PDXs was assessed using WGS and WES. Ninety-two % of the mutations with an allele frequencies (AF) >30% were conserved. Of altogether 609 SNVs and InDels (AF>10%) detected in primary samples 68 % (417) were also detected in the corresponding PDXs. In some PDXs, mutations not identified by targeted sequencing in the primary sample (NOTCH1, PHF6, DNA11, USP9X) occured at a high AF (22-59%), which may have been the result of selection of a subclone or of an acquisition of the mutation in the process of PDX generation.Thirty of 35 (86%) large CNA assessed by WGS were preserved in corresponding PDX models indicating that CNAs are likely earlier events in the leukemogenesis that are present at high AF in the primary samples. CNA analysis with MLPA confirmed that PDXs retained 96% (80/83) of the deletions and amplifications of primary leukemia.In 18 out of the matched 22 PDXs all clonal mutations (AF >30%) detected in the primary sample have been preserved in the PDX. Seventeen out of 22 contained more than 1 clone. Fifteen out of these 17 retained the complexity of primary sample with different subclones successfully engrafted.When considering the 11 matched pairs, we observed 4 different engraftment types of initial disease. Type 1 engrafted in a similar way in which they relapsed preserving all or nearly all mutations found in patients' sample (4 samples). Type 2: the relapsing clone that was not detected in initial diagnosis was present in PDX derived of initial diagnosis (PDX_INI) but it was not dominant (3). Type 3: the relapsing clone that was present at initial diagnosis did not engraft into PDX_INI (2). Type 4 preserved the relapsing clone as well as the mutations specific for initial diagnosis (2). Out of 11 PDXs of relapse 9 preserved all or nearly all mutations detected in primary sample. In 2/11 PDXs of relapse one of the clones preexisting in primary relapse was selected for, while the other one was lost.Differential methylation analysis by EPIC array of 21,711 promoters of primary samples and PDXs revealed that the average methylation in primary samples (β=0.43) did not differ from that in PDXs (β=0.45). Principal Component Analysis and unsupervised hierarchical clustering showed that matched pairs, both of the primary and the PDX samples cluster together (ρ=0.9912). Similarly, analysis of chromatin accessibility by ATAC-seq showed that more than 92% of peaks of a height > 20 and more than 75% of the peaks of a height of > 10 were preserved between primary samples and corresponding PDXs.In conclusion, PDX models largely reflect the heterogeneity and the complexity of the primary leukemic sample at the genomic and epigenomic level, although some clonal and particularly subclonal mutations can be lost. Differences in engraftment and various engraftment scenarios of initial diagnosis in contrary to relapse samples suggest that relapse samples are more preselected and stable in engraftment. Altogether, this overall genomic and epigenomic stability of the primary T-ALL in the PDX renders the models as a suitable source for material when primary samples are limiting and also to reduce uncontrollable pre-analytical changes in primary samples. Further, these models are suitable for the preclinical development of new treatment strategies for pediatric T-ALL. DisclosuresSchrappe:Baxalta: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Medac: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding.

Tyrosine Kinase Inhibitor-Induced Defects in DNA Repair Sensitize FLT3(ITD)-Positive Acute Myeloid Leukemia Quiescent and Proliferative Cells to PARP Inhibitors

Mutations in the FMS-like tyrosine-kinase 3 (FLT3) such as internal tandem duplications (ITD) can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors, which are not curative. Poly (ADP-ribose) polymerase (PARP) inhibitors have been recently successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying deficiency in a major DNA double strand break (DSB) repair pathway - homologous recombination (HR). Using personalized medicine approach we recently reported that a cohort of AMLs displayed deficiencies in HR and also in another major DSB repair pathway - non-homologous end-joining (NHEJ) (Nieborowska-Skorska et al., J.Clin.Invest., 2017). HR and NHEJ play a key roles in DSB repair in proliferating (HR and NHEJ) and quiescent (NHEJ) cells, and HR/NHEJ deficiency sensitized quiescent and proliferating AML stem cells to synthetic lethality triggered by PARP inhibitors olaparib and BMN673 (talazoparib). Although AML cells expressing FLT3(ITD) displayed activation of HR and NHEJ activities, we show here that inhibition of FLT3(ITD) kinase by tyrosine kinase inhibitor AC220 caused downregulation of selected DNA repair proteins such as BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in simultaneous inhibition of HR and NHEJ. PARP inhibitors olaparib and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells thus mimicking synthetic lethality. In addition, combination of FLT3 inhibitor and PARP inhibitor in vitro eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells and leukemic progenitors from human and mouse leukemia samples. Moreover, AC220 combined with BMN673 were highly effective in vivo against FLT3(ITD)-positive primary AML xenograft cells, including leukemia initiating cells. In conclusion, we postulate that FLT3 inhibitor-induced deficiencies in HR and NHEJ sensitized FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP inhibitor. Therefore FLT3(ITD) could be used as a precision medicine marker identifying patients with AMLs, which may benefit from therapeutic regimen combining FLT3 and PARP inhibitors. DisclosuresKoschmieder:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Roche: Other: Clinical Trial participation. Valent:BMS: Honoraria; Deciphera: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Blueprint: Research Funding; Celgene: Honoraria, Research Funding; Pfizer: Honoraria; Teva: Honoraria; Ariad: Honoraria, Research Funding; Incyte: Honoraria.

Sy-1365, a Potent and Selective CDK7 Inhibitor, Exhibits Anti-Tumor Activity in Preclinical Models of Hematologic Malignancies, and Demonstrates Interactions with the BCL-XL/BCL2 Mitochondrial Apoptosis Signaling Pathway in Leukemia

Introduction: The transcriptional kinase CDK7 has been implicated in the pathogenesis of multiple malignancies, including hematologic malignancies, and may play important roles in regulation of oncogenic transcriptional dependencies and in regulation of the mitochondrial apoptosis machinery in tumors. SY-1365, a selective inhibitor of CDK7, was developed to exploit tumor dependencies driven by CDK7 and is in clinical development in patients with advanced solid tumors. We have previously reported that SY-1365 selectively induces apoptosis in leukemic cells relative to non-malignant cells in vitro, and demonstrates anti-tumor activity in AML xenografts. Here we report that SY-1365 has potent inhibitory activity against a broad panel of hematologic malignancies in vitro and ex vivo, induces regressions in AML xenografts using a clinically relevant dosing regimen, and demonstrates synergy with the BCL2 inhibitor venetoclax in AML cells in vitro.Methods: SY-1365 dose-response curves were measured using the ATP-lite assay in a panel of 85 leukemia and lymphoma cell lines in vitro, and in a panel of 226 patient-derived tumor samples from 5 leukemic indications cultured ex vivo. In vivo tumor inhibitory activity was evaluated using once weekly (QW) and twice weekly (BIW) dosing regimens in ML-2 and Kasumi-1 AML xenografts. Growth-rate (GR) adjusted dose response curves were used to classify cell lines into low and high sensitivity groups, and were evaluated relative to RNA expression data to identify markers predictive of sensitivity to SY-1365. Synergy between SY-1365 and venetoclax was evaluated in vitro using KG-1 AML cells.Results: In 42 leukemia lines, SY-1365 had a median IC50 of 11nM (range 1-79nM), with 48% (20/42) demonstrating particularly low IC50s (<10nM). In 43 lymphoma lines, the median IC50 was 6nM (range 1-39nM), with 59% (27/46) demonstrating low IC50s (<10nM). Similarly, SY-1365 demonstrated potent inhibitory activity against patient-derived leukemia samples cultured ex vivo (median IC50 of 28nM), with 82% (185/226) of the samples showing IC50 below 100nM and 8% (19/226) under 10nM. Furthermore, analysis of growth rate (GR) in hematologic tumor cell lines showed that <10nM SY-1365 induced cytotoxicity (GR<0) in 19% (8/42) of leukemia and 21% (9/43) of lymphoma lines. To evaluate SY-1365 anti-tumor activity in vivo, 2 AML cell lines that demonstrate cytotoxic responses to SY-1365 in vitro (Kasumi-1 and ML-2) were grown as subcutaneous xenografts in mice. QW and BIW dosing of 40mg/kg SY-1365 induced tumor regressions in Kasumi-1 xenografts, and BIW dosing induced tumor stasis in ML-2 xenografts. Tumor growth inhibition was detected with a 20mg/kg BIW dosing regimen in both models. Comparison of SY-1365 sensitivity with RNA expression in hematologic tumor cell lines revealed that lower expression of BCL2L1, which encodes the mitochondrial apoptosis inhibitor BCL-XL, was highly predictive of sensitivity in leukemia cell lines (Accuracy=89%, FDR<0.05); no such relationship was observed in lymphoma lines. Considering the established role of BCL2 and the mitochondrial apoptosis pathway in leukemia, we reasoned that BCL-XL low-expressing leukemias, in addition to being sensitive to SY-1365, would also be reliant on BCL2 for survival. Indeed, in vitro combination studies demonstrated synergistic inhibitory activity of SY-1365 with the BCL2 inhibitor venetoclax in KG-1 AML cells. A potential synergistic interaction with SY-1365 and venetoclax is currently being explored in vivo with AML xenografts.Conclusions: SY-1365 shows potent in vitro inhibitory activity in multiple hematological indications and can induce AML xenograft tumor regression in mice. In vitro exploratory biomarker and tumor cell inhibitory combination studies in AML suggest a role for mitochondrial apoptosis signaling in mediating sensitivity to SY-1365 and support the evaluation of a combination with venetoclax. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors (NCT03134638). The results described here support the potential for additional exploration in patients with hematological malignancies. DisclosuresHodgson:Syros Pharmaceuticals: Employment, Equity Ownership. Johannessen:Syros Pharmaceuticals: Employment, Equity Ownership. Rajagopal:Syros Pharmaceuticals: Employment, Equity Ownership. Hu:Syros Pharmaceuticals: Employment, Equity Ownership. Orlando:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. McKeown:Syros Pharmaceuticals: Employment. Tyner:Takeda Pharmaceutical Company: Research Funding; Agios Pharmaceuticals: Research Funding; Incyte Corporation: Research Funding; Gilead: Research Funding; Janssen Pharmaceutica: Research Funding; Syros: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; AstraZeneca: Research Funding; Leap Oncology: Consultancy. Sprott:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Di Tomaso:Syros Pharmaceuticals: Employment.

RNA Editing By APOBEC3A Confers Dynamic Mutational Load in Monocytic Leukemias

Background: Although genomic mutations play an important role in the initiation or progression of myeloid leukemias, the role of mutations at the transcript level is less well understood. Myeloid neoplasms have recurrent somatic mutations in genes for pre-mRNA splicing, cohesin complex, histone modification, and DNA methylation. However, the number of somatic mutations identified in myeloid neoplasms (average 9-13) is usually lower than that of solid tumors. Recent analyses of baseline transcriptome sequences in various cancers have revealed abundant adenosine to inosine (A>I) RNA editing events, mainly at intronic and untranslated regions of genes. The role of condition-specific RNA editing in cancer, especially of the cytosine to uracil (C>U) type, however, remains essentially unknown. We recently discovered antiviral restriction enzyme APOBEC3A (A3A) cytidine deaminase as a novel cellular site-specific C>U RNA editing enzyme in monocytes. RNA editing by A3A is dynamically induced by interferons and/or severe hypoxia in monocytes, resulting in stop codon gains or missense changes in transcripts of scores of genes. The effect of hypoxia in inducing RNA editing by A3A can be mimicked by inhibition of mitochondrial respiration. Whether A3A-mediated RNA editing occurs in monocytic leukemias, including acute monocytic leukemia (AMoL) and chronic myelomonocytic leukemia (CMML), is unknown. We hypothesize that dynamic C>U RNA editing by A3Acontributes to mutational load of leukemic cells under stressful conditions of hypoxia and interferon exposure. In this study, we examined transcript sequences of monocytic leukemias for evidence of inducible RNA editing. We also examined the role of mitochondrial respiratory inhibition, which induces RNA editing by A3A, in growth of leukemic progenitors of CMML.Methods: Monocytic leukemia samples are obtained from the RPCI Leukemia Bank. Standard diagnostic information included classification (WHO 2008 and FAB), fraction of monocytic component (mature monocytes, promonocytes or monoblasts by flow cytometry and/or aspirate differential count), cytogenetic analyses (conventional karyotype and targeted FISH), and survival data. Leukemic BM mononuclear cells (BM-MNC) prepared by the Ficoll gradient method are thawed and cultured in hypoxia (1% O2) with interferon type 1 (IFN-1). DNA and RNA are isolated. AMoL samples are subjected to nextgen high throughput exome and RNA sequencing. Once variants from DNA and RNA of corresponding samples are obtained, the set difference of variants is extracted to keep those detected on RNA samples. We also examined 3 CMML bone marrow samples for evidence of hypoxia-inducible RNA editing by analyzing selected RNA editing sites. Colony forming unit (CFU) assays are also performed in CMML samples.Results: We find that the A3A gene is robustly expressed inboth in AMoL and CMML. Hypoxia (with or without IFN-1) induced A3A-mediated RNA editing in AMoL and CMML samples. We previously showed that several RNA edited genes by A3A in normal monocytes function in recurrently altered pathways in myeloid leukemias. Sanger sequencing confirmed RNA editing of such genes in monocytic leukemia samples as well (Table 1), suggesting that epitranscriptomic sequence changes may contribute to molecular defects driving leukomogenesis. RNA seq analysis of AMoL samples (n=3) showed that dozens of genes acquire nonsense or missense mutations at the transcript level under the conditions of severe hypoxia and IFN-1. CFU assays of CMML samples showed that inhibition of mitochondrial respiration, which triggers A3A-mediated RNA editing, inhibits the growth of progenitor cells (Figure 1).Conclusions: We show that A3A-mediated RNA editing is active in monocytic leukemias and targets genes functioning in pathways recurrently altered in myeloid leukemias. RNA editing by A3A may contribute to growth arrest and survival of leukemic progenitors under certain stress conditions. To our knowledge, these results provide the first experimental evidence that conditional RNA editing by A3A confers substantial mutational load in myeloid leukemias. Such RNA editing events will dynamically increase the total mutational load in monocytic leukemias, and may create neo-antigens for immune therapy by checkpoint inhibition. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Lineage Deconvolution Using Chromatin Accessibility and DNA Methylation Shows a Spectrum of Lineage Characteristics and Differentiation States across Acute Leukemias

Hematopoietic differentiation is characterized by the organized epigenetic reprogramming of hematopoietic stem cells to terminally differentiated cells. Acute leukemias represent a disruption of this process and are categorized into myeloid and lymphoid subtypes on the basis of surface marker patterns identified by flow cytometry. Biphenotypic and other poorly specified leukemias such as early T-cell precursor acute lymphoblastic leukemias (ETP T-ALLs) highlight the limitations of this technology. Here we describe a novel approach to characterize leukemia lineage on the basis of epigenetic characteristics. Using model-based deconvolution, we show that DNA methylation profiling and ATAC-seq chromatin accessibility maps can be used to specify leukemia lineage characteristics more precisely. On in-vitro mixtures of purified cells, it can quantitatively determine sample composition with R2 = 0.89. Extending this method to evaluate primary leukemia samples, we show that ETP T-ALLs exhibit a mixture of lineage characteristics that varies by patient. Typical T-ALL patient samples exhibit predominantly lymphoid characteristics most similar to the common lymphoid progenitor (CLP) stage. In contrast, ETP T-ALL cases exhibit a mixture of myeloid and lymphoid features, with some showing dominant CLP features, some a mix of both CLP and granulocytic macrophage progenitor (GMP) characteristics, and one with dominant GMP features. Biphenotypic leukemias also contained CLP dominant, GMP dominant, and mixed cases. CEBPa-silenced AMLs, which had previously been described as exhibiting a T-cell like phenotype, exhibited dominant CLP characteristics in contrast to other AMLs that exhibit dominant GMP features. In the future, we hope to adapt this method to assist in determining whether myeloid or lymphoid directed therapy should be used for leukemias with ambiguous lineage characteristics. [Display omitted] DisclosuresLevine:Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Epigenetic and Transcriptomic Profiling of MLL Rearranged Infant Acute Lymphoblastic Leukemia

[Background] Infant acute lymphoblastic leukemia (ALL) accounts for less than 5% of childhood leukemia, and is characterized by Mixed lineage leukemia (MLL also called KMT2A ) rearrangements found in approximately 80% of patients. Also found in 5-10% of ALL in older children and adults, MLL -rearrangement positivity is highly associated with poor outcome in ALL; there is an urgent need for molecular findings of potential therapeutic targets and/or risk stratification markers, which can be translated into the clinical practice. Recently, the mechanisms that MLL fusion proteins recruit epigenetic regulators, including histone H3 lysine 79 methyltransferase DOT1L and BET family histone readers, to activate the transcription of target genes have been reported, suggesting epigenetic dysregulation should play a central role in the leukemogenesis. However, DNA-level methylation profiles of MLL -rearranged leukemia and their associations with gene expression profiles are not fully characterized.[Materials and Methods] Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. Both methylation array and RNA sequencing were performed on the diagnostic leukemia samples from 38 infants with ALL, consisting of 30 cases with MLL -rearrangements identified by fluorescence in situ hybridization or Southern blotting, and 8 MLL -germline cases. Methylation beta values were normalized with beta-mixture quantile normalization method. For RNA sequencing data analysis, we used publicly available Genomon Pipeline.[Results] RNA sequencing confirmed MLL -rearrangements in 30 cases, whose partner genes comprised AFF1 (n=15), MLLT1 (n=6), MLLT3 (n=3), MLLT10 (n=2), EPS15 (n=1), AFF2 (n=1), ABI1 (n=1), and SARNP (n=1). As expected, DNA methylation-based hierarchical clustering divided the cases into 2 clusters with highly distinctive methylation profiles: MLL -rearranged cluster (MLL -R cluster) and MLL -germline cluster (MLL -G cluster). Intriguingly, 3 out of 30 (10%) MLL -rearranged cases were classified into MLL -G cluster; these cases revealed uncommon partner genes, such as ABI1 , SARNP , and MLLT10, respectively. ABI1, an Eps8 SH3 domain-binding protein, and SARNP, a SAP domain containing ribonucleoprotein, are rare fusion partners of MLL. The MLL -germline leukemia-like methylation profiles suggested different leukemogenic mechanisms from common fusion partners, such as AFF1 and MLLT1, which directly or indirectly interact with DOT1L and positive transcription elongation factor b. Because DOT1L-binding domain of MLLT10 was retained in both of the 2 cases with MLL-MLLT10 in our cohort, additional mechanisms causing MLL-germline leukemia-like methylation in a single case may be present. As a result of inter-cluster comparison, MLL -R clusterexhibited global hypermethylation compared to MLL -G cluster (P -value < 2.2 × 10−16, Kolmogorov-Smirnov test). Methylation-based gene set enrichment analysis demonstrated significant promoter hypermethylation on cell development/differentiation-related gene sets and hypomethylation on DNA replication/cell proliferation-related gene sets (P -value < 0.01, hypergeometric test) in MLL -G cluster. Consistently, RNA sequencing-based gene expression analysis demonstrated overexpression of cell cycle regulators, such as cyclins/cyclin-dependent kinases, in MLL -R cluster, which may be associated with more aggressively proliferating diseases.[Conclusion] Corresponding to known aberrant histone modifications in MLL -rearranged leukemia, largely different DNA methylation profiles between MLL -R and MLL -G clusters have been demonstrated. Because DNA-level methylation on CpG dinucleotides interactively affects histone modifications, these results implicated aberrant DNA methylation in the pathogenesis of MLL -rearranged leukemia as a part of epigenetic dysregulation. We also identified a subset of MLL -rearranged cases with MLL -germline leukemia-like methylation/expression profiles, indicating that not only MLL -rearrangement positivity, but also the types of MLL -rearrangement partners may affect a biological feature of leukemic cells. These cases may possibly be therapeutically stratified as relatively lower risk group compared to other MLL -rearranged cases. Further detailed analyses using clinical information are planned. DisclosuresNo relevant conflicts of interest to declare.

PD-1 Inhibition Enhances Blinatumomab Response in a UCB/PDX Model of B-Cell Acute Lymphoblastic Leukemia

IL-2Ry immune deficient mice engrafted with human umbilical cord blood (UCB) develop human immune systems with mixed B, T, NK, and myeloid populations. We have previously modified NOD/SCID/Gamma (NSG) and NOD/RAG/Gamma (NRG) mice to include transgenic expression of human cytokines SCF, GM-CSF, and IL-3 (NSGS/NRGS). Relative to NSG/NRG, NSGS/NRGS mice have increased myeloid and NK fractions, more complete B cell differentiation, and faster T cell reconstitution. Additionally, both innate and adaptive immune function is also improved as measured by DTH assay and human antigen specific antibody production in response to immunization. NSGS and NRGS mice also more readily engraft leukemia samples, particularly in the absence of conditioning. We took advantage of the improved nature of hematopoietic xenografts in NRGS mice to determine whether or not humanized mice can serve as an in vivo model to test immune therapies against human leukemia.Busulfan conditioned NRGS mice were humanized by engraftment with red blood cell depleted UCB. Engraftment was monitored by the appearance of significant levels of human CD45+CD3+ T cells in the peripheral blood (PB). Once mice were reconstituted with >25% T cell graft in PB, we injected an aggressive, relapsed/refractory t(4;11) patient sample to induce fatal ALL in the humanized NRGS mice. All control humanized NRGS mice succumbed to disease between 40 and 60 days after leukemia injection with rapidly expanding B-ALL in the PB, bone marrow, and spleen. Mice treated with several courses of the bi-specific T cell engager (BiTE) molecule, blinatumomab, were partially protected from disease, and only half of these mice developed detectable B-ALL at the end of the experiment (day 90 post-injection). Strikingly, the combination of blinatumomab and the PD-1 antibody, pembrolizumab, fully protected the mice from B-ALL disease. All blinatumomab/pembrolizumab treated mice survived until the end of the experiment at which time there was no evidence of disease in PB, bone marrow, or spleen preparations. Importantly, additional experiments showed that this blinatumomab effect was dependent on the presence of the human immune system. Blinatumomab did not affect initiation (early treatment) or progression (late treatment) of B-ALL in non-humanized mice that were engrafted with the same patient sample.T cell levels were monitored during the experiment and showed a slight decline upon blinatumomab treatment. In contrast, T cell levels were significantly higher in mice that received both blinatumomab and pembrolizumab, implying augmented T cell activation in vivo. This may be a relevant finding because it is likely that some patients fail blinatumomab therapy due to sub-optimal T cell levels. The addition of pembrolizumab may offer means to combat this problem. Nevertheless, mice with less than 20% CD45+CD3+ T cells in the PB failed to respond to either blinatumomab or combined blinatumomab/pembrolizumab treatment, indicating there is a lower limit to potentiating the T cell response during BiTE therapy.These results demonstrate the potential utility of dual UCB/PDX models for examining novel combinations of immune therapies in vivo and suggest that additional studies combining BiTEs with immune checkpoint inhibitors are warranted. DisclosuresNo relevant conflicts of interest to declare.

An Enhanced Zebrafish Xenograft Model to Study the Behavior of Human Hematopoietic Stem Cells and Patient Derived Leukemia In Vivo

Human hematopoietic stem cells (HSCs) are characterized by their unique ability to self-renew and differentiate into multiple cell types. Transplantation of human HSCs into immunocompromised mice is the gold standard for evaluating the in vivo repopulation capacity of these cells and has been adapted to evaluate the leukemic potential of leukemia-initiating cells (LICs) in acute leukemia. In spite of the popularity of this approach, certain cellular subsets do not survive post xenotransplantation as they are dependent on human factors that may be missing in the murine microenvironment. We pioneered human leukemia xenografts in the zebrafish model (Corkery et al., 2011; Bentley et al., 2015) as a parallel strategy, taking advantage of the immuno-permissiveness and transparency of zebrafish larvae to provide unprecedented continuous observation of leukemia evolution. However, as in mice, the addition of human cytokines and growth factors may further improve engraftment and optimize the utility of the zebrafish as a preclinical transplant model for medium to high-throughput therapeutic screening by creating a microenvironment more comparable to that found in patients.Using transgenic technology, we created novel zebrafish lines that express human stem cell factor (SCF/KITLG), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), and stromal cell-derived factor 1 alpha (SDF1α/CXCL12). All of these factors are essential for the survival and expansion of HSCs, progenitors and LICs. CMK, GRWi (Down Syndrome- acute myeloid leukemia (DS-AML)) and Jurkat (T-acute lymphoblastic leukemia) cell lines were injected into the yolk sac of these multi-cytokine zebrafish embryos. These cells engrafted and exhibited increased cell proliferation and physiological migration compared to control casper embryo transplants. Moreover, only in the multi-cytokine fish did these cells home to hematopoietic niches, including the caudal hematopoietic tissue (CHT) (equivalent to the fetal liver) at 7 days-post injection (dpi) and the kidney marrow at 11 dpi.The yolk sac is not the physiological site for hematopoietic cell growth and thus we injected patient-derived AML cells into the circulation of the multi-cytokine fish. We observed similar trends in proliferation and migration as seen with leukemia cell lines. Historically, human HSCs do not survive beyond 24h in zebrafish xenografts (Pruvot et al. 2011). Given our success with patient-derived leukemia samples, we wanted to see if our multi-cytokine fish would provide a more favorable environment for human HSC survival. Indeed, primary human umbilical cord blood-derived HSCs were injected into the circulation of zebrafish larvae and survived past 3 dpi in the multi-cytokine fish but not in casper control larvae.Transgenic zebrafish expressing human cytokines provide an enhanced model for studying human leukemia and stem cell biology in a visually tractable system that more closely recapitulates the human microenvironment. The inherent capacity of the zebrafish for rapid and efficient chemical screening will be exploited in this model for preclinical testing of anti-leukemic therapies and to evaluate HSC enhancing agents in vivo . DisclosuresBerman:AGADA Therapeutics: Research Funding.

Over-Expression of the Local Bone Marrow Renin-Angiotensin System in Acute Myeloid Leukemia.

Local bone marrow (BM) renin-angiotensin system (RAS) is an autocrine-paracrine system affecting hematopoiesis [Haznedaroglu IC, et al. A local RAS in the bone marrow. MedHypotheses. 1996; 46:507–10., Haznedaroglu IC. A local RAS in the bone marrow still awaits its Christopher Columbus. ExpHematol. 1998; 26:279., Haznedaroglu IC, et al.Towards the understanding of the local hematopoietic bone marrow RAS. IntJBiochemCellBiol. 2003; 35:867–80.] Angiotensin II type 1a (AT1a) receptors are present on the CD34+ hematopoietic stem cells from the BM or cord blood (CB) [Goker H, et al. Local umbilical cord blood renin-angiotensin system. AnnHematol . 2005; 84:277–81]. Angiotensin II stimulates the proliferation of BM and umbilical CB hematopoietic progenitors. Local RAS may also be involved in leukemogenesis and polycythemia vera [Aksu S, et. al. Enhanced Expression of the Local Haematopoietic Bone Marrow Renin-Angiotensin System in Polycythemia Rubra Vera J Int Med Res (in press), Aksu S, et. al. Over-expression of angiotensin converting enzyme (CD143) on leukemic blasts as a clue for the activated local bone marrow RAS in AML. Leukemia and Lymphoma (in press)]. Angiotensin converting enzyme (ACE) hyperfunction may lead to the acceleration of negative hematopoietic regulator peptide, AcSDKP, metabolism, which in turn lowers its level in the bone marrow microenvironment, finally removing the anti-proliferative effect of AcSDKP on the hematopoietic cells and blasts. Renin expression could have a role in leukemia development and angiotensin may act as an autocrine growth factor for AML cells. In this study, the expression of the mRNAs of the major RAS components, namely ACE, renin, and angiotensinogen in human BM samples were quantified by RT-PCR to confirm the presence of the local BM RAS. Furthermore, we assessed quantitative alterations of the ACE, renin, and angiotensinogen mRNAs in leukemic BM samples. The aim of this study was to search those major RAS components in the leukemic blast cells taken from the BM of the patients with AML. BM aspirations were obtained from 10 patients with AML and 8 patients with non-malignant hematological disorders. All of three gene expressions were found to be significantly higher in the BM samples of AML patients compared to the BM samples of the control group (Figure 1). Pathological activity of the local RAS-mediated regulation may be important, since the angiotensin peptides represent a molecular target in the disease management. [Display omitted]

TRAIL-R3 Expression on Myeloid Leukemic Blasts Is Related to a Shortened Overall Survival.

Since chemotherapy and transplantation can cure only around 35% of patients with acute myeloid leukemia (AML), there is still need for complementary and targeted treatment modalities. One of them could be the use of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL is expressed by effector T cells and induces apoptosis via the death receptor intrinsic pathway. Activation of this pathway in addition to the mitochondrial pathway (by chemotherapy) has synergistic effects in vitro. In human, 4 membrane bound receptors have been identified: two of them (R1 and R2) contain a functional death domain and are capable of starting the apoptotic cascade, and two others (R3 and R4) lack a functional death domain and function as decoy receptors. Most normal cells express R3 and R4, where many tumor cells express R1 and R2. This makes soluble recombinant TRAIL an attractive candidate for targeted therapy; phase 1 clinical studies for solid tumors are launched. Until now, sparse data on TRAIL sensitivity of myeloid leukemic cells have demonstrated low TRAIL sensitivity. We investigated blood and bone-marrow samples of 113 patients with AML for TRAIL receptor expression by flow-cytometry. In contrast with published data, we found presumably (pro-apoptotic) R1 and R2 expression (mean percentage positive cells 16% and 34%, range 0–79% and 0–97% respectively) versus R3 and R4 expression (mean 9% and 10%, range 0–71% an 0–45%) indicating a TRAIL sensitive profile for myeloid blasts. Surprisingly, the expression of the anti-apoptotic R3 strongly correlated to survival. Expression of > 25% blasts positive for R3 resulted in shortened overall survival (p=0.0051), see figure 1. In multivariate analysis R3 expression remained a significant prognostic factor next to cytogenetics (p=0.03 and p= 0.015 respectively). In vitro studies on 4 myeloid leukemic cell lines confirmed TRAIL sensitivity in cell lines that expressed R1 and R2. Furthermore, simultaneous expression of R3 clearly reduced the amount of apoptosis, suggesting that TRAIL effects are inhibited by binding to R3. TRAIL induction studies on fresh leukemic samples are currently underway in our laboratory.In conclusion, our data suggest that, in contrast to earlier reports, there might be a role for TRAIL in apoptosis induction of AML blasts. R3 expression is a strong predictor for overall survival and modulation of R3 might yield additional new therapeutic options for AML patients. [Display omitted]

Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small molecule inhibitors with a well-defined mechanism of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary AML samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.