Fusion Partner Research Articles

The Methylome Atlas of Acute Leukemia Enables Novel Clinical Diagnostic Tool for AML

In 2022, both the World Health Organization (WHO) and the European LeukemiaNet (ELN) highlighted the formidable challenges of categorizing certain Acute Leukemia subtypes, such as AML with rare recurring translocations, complex karyotypes, not otherwise specified, and myelodysplastic-like entities. As characterizations of new variants and fusion partners become more prevalent, it is difficult to assess which ones, alone or in combination, generate which leukemia phenotypes. To that end, WHO posed methylation profiling as “the technology at present best suited for addressing lineage and thereby the cell population of origin of tumors” (PMID: 34921008), which invites an opportunity to evaluate the DNA methylome as a clinical diagnostic avenue in hematopoietic malignancies like AML. Thus, we hypothesize that the DNA methylome holds promise in further refining AML classification. In this study we describe the assembly of the largest publicly available methylation dataset of acute leukemias so far, which combines 11 high-quality clinical trials/studies: NOPHO ALL92-2000 (n=796), AAML0531 (n=628), AAML1031 (n=581), BeatAML (n=316), TCGA AML (n=194), French GRAALL 2003-2005 (n=153), TARGET ALL (n=131), CETLAM SMD-09 (n=83), AAML03P1 (n=72), Japanese AML05 (n=64), and CCG2961 (n=41), resulting in a total of 3,059 subjects after preprocessing. Samples were obtained either from bone marrow or peripheral blood, with DNA methylation (meDNA) data procured using the Illumina methylation array 450k or EPIC array, which share 452,453 probes with same chemistry and design. To independently validate the findings derived from the discovery cohort, we processed in parallel meDNA array data from bone marrow specimens at diagnosis of AML patients treated on the multi-center clinical trials AML02 (n=159) and AML08 (n=42) led by St. Jude Children's Research Hospital. Processing the raw data followed best practices from the literature using SeSAMe (PMID: 30085201, 27924034). To generate the atlas, we used a novel dimensionality reduction unsupervised learning algorithm called Pairwise Controlled Manifold Approximation (PaCMAP), which allowed compression of 319,738 processed CpG values into two components for visualization (Figure 1) and five components for downstream classification analysis. To empirically assess classification accuracy of PaCMAP results, we implemented a machine learning pipeline with hyperparameter tuning, 10-fold cross validation, and assessed accuracy per class in discovery and validation cohorts. Not all samples, however, had clinical diagnostic annotation available in the dataset, so only those with the annotations were used in the supervised machine learning model (n=1399 in discovery and n=110 in validation). Our resulting atlas unveils several clusters of samples that defined 6 hematopoietic lineages: AML, ALL, MDS-related or secondary myeloid neoplasms (MDS-like), Acute promyelocytic leukemia (APL), mixed phenotype leukemia, and otherwise-normal control. These are further subdivided into 30 subclasses overlapping with WHO 2022 and ELN 2022 clinical diagnostic annotations. Subsequent analyses comparing methylation-based subtype prediction to clinically annotated subtypes revealed an overall concordance score of 0.936, with per class 10-fold CV concordance ranging from 0.82 for MDS-like to 1.00 in APL and NUP214 fusions. Importantly, the validation (test) cohort showed accuracies per class of 0.91 for AML with KMT2A-r (n=47) and 1.00 for AML rare recurring translocations (n=10). These are subtypes with large genomic heterogeneity that may be better represented at the epigenomic level. Additionally, these are largely considered standard to high-risk groups with poor prognosis, which invites further studies aiming at uncovering the biological mechanisms behind the methylation patterns governing this classification. Finally, the resulting classifier allowed prediction of WHO/ELN clinical diagnosis for 91 samples in the validation cohort that were previously categorized as “Normal Karyotype”, “Other” or blank. In conclusion, our study effectively showcases the use of a methylome atlas in enhancing the diagnosis of AML subtypes.

Clinical Characteristics, Genetic Profile and Prognosis of Adult Acute Myeloid Leukemia with NUP98 Rearrangement

Introduction: Nucleoporin 98 ( NUP98) rearrangement has been proposed in 2022 as one type of acute myeloid leukemia (AML) with defining genetic abnormalities. It occurred more often in pediatric leukemia with poor outcome. However, in adult AML with NUP98 rearrangement, patients may show different clinical characteristics, genetic profile and outcome, as we reported here. Method: We retrospectively collected and analyzed the data of adult AML patients with NUP98 rearrangement who were diagnosed at Shanghai Institute of Hematology from May 2019 to December 2022, including clinical characteristics, laboratory examination, genetic abnormalities, treatment strategies and outcome. All patients underwent next generation sequencing and RNA sequencing. Result: A total of 21 AML patients with NUP98 rearrangement were included in this study, counting for 2.3% of all AML patients (930 cases). The median age was 40 (19-64) years old. The median white blood cell and platelet count was 31.1 (1.5-313.6) and 48 (15-140) ×10 9/L, respectively. Interestingly, 11 (52.4%) patients suffered from severe or fatal hemorrhage irrelative with platelet count or coagulative function, including 4 cerebral bleeding (3 died), 1 urgent uterine bleeding, 1 gastro-intestinal bleeding, 1 retina bleeding and 4 massive bleeding of subcutaneous soft tissue or mucous membranes. The FAB types involved M2, M4 and M5. Eight patients carried normal karyotype, 6 were found with t(7;11)(p15;p15), all of whom had NUP98::HOXA9 fusion gene, 2 with +8 and 1 complex karyotype. According to ELN 2022 risk classification, 14 (66.7%) were at intermediate risk and 6 (28.6%) at adverse risk, 1 unclassified. Five fusion partners with NUP98 were detected: NUP98::NSD1 (11 cases), NUP98::HOXA9 (7 cases), NUP98::HOXA11, NUP98::HMGB3 and NUP98::TNRC18 (each with 1 case). Of which NUP98::TNRC18 was first reported by our center (see Cheng WY, et al. Proc Natl Acad Sci U S A. 2022;119(49):e2211429119). NUP98 rearrangement also co-existed with several mutations. FLT3-ITD (12 cases, of which 10 were co-existed with NUP98::NSD1) and WT1 (7 cases) were most frequently occurred. Patients with relapse/refractory disease had more additional mutations than de novo status (Figure 1). Nineteen patients were treated with standard dose of “7+3” induction therapy, and 2 with hypomethylating agents (HMA) plus venetoclax (VEN). Complete remission (CR) was achieved by 8 (38.1%) patients after first induction, 2 (9.5%) early deaths were both due to cerebral hemorrhage. Salvage induction varied from intensive chemotherapy (IC, including “7+3” and CLAG±M) to low-intensity therapy (LIT, including priming chemotherapy±HMA and HMA+VEN). FLT3 inhibitors (FLT3i) were added to 5 patients carrying FLT3-ITD, and they all achieved CR. A total of 15 (71.4%) patients achieved CR after 1-2 courses of induction (8 mainly benefited from IC, 7 mainly benefited from LIT±FLT3i), 1 survived from allogeneic hematopoietic stem cell transplantation (allo-HSCT) without reaching remission before, and 3 died from refractory disease within 6 months. Three patients relapsed after CR, 2 died without allo-HSCT, and the other one survived after CR2 and sequential allo-HSCT. A total of 14 patients underwent allo-HSCT after CR, 7 were measurable residual disease (MRD) negative and 7 were positive by flow cytometry before HSCT, none of them relapsed till the last follow-up. With a median follow-up time of 19.2 months, the median overall survival (OS) and event-free survival (EFS) were not reached, estimated 2-year OS and EFS rate were 66.7% and 57.1%, respectively. For patients achieving CR or not, the median OS were not reached vs 3.2 months (95% CI 0-8.361, P<0.001, Figure 2). Ten patients were diagnosed with NUP98::NSD1 co-occurring with FLT3-ITD (once considered as unfavorable prognosis), ending up with a CR rate of 70%, and a 2-year OS and EFS rate of 80% and 60%, respectively, making no significant difference compared to others ( P=0.236 and 0.725, respectively). Conclusion: Adult AML with NUP98 rearrangement may combine with a set of partner fusion genes and mutations, with NUP98::NSD1 and FLT3-ITD as the most common combination. Both IC an LIT can induce CR, and FLT3i will benefit those with FLT3 mutations, also prevention of fatal bleeding should be paid attention to. After achieving CR with whatever method and whatever MRD status, allo-HSCT is the key to long-term survival.

JAK2 Kinase Fusion and Myeloid Involvement in Acute Lymphoblastic Leukemia

Introduction JAK2 fusion genes (FGs) are rare but can be found in various phenotypes of myeloid or lymphoid malignancies. Both in the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors (WHO-HAEM5, PMID: 35732831) and the International Consensus Classification (ICC, PMID: 35767897), JAK2-FGs were included in the diagnostic entity of myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK or MLN-eo), and both suggest MLN-TK/eo as a priority diagnostic entity. It is highlighted in ICC that cases presenting as acute lymphoblastic leukemia (ALL) with M/LN-eo differ from de novo B-ALL and T-ALL in the involvement of myeloid cells. There is still a lack of research reports about how many JAK2-FG-positive ALL cases developed from M/LN-eo or had myeloid involvement. Methods A total of 21 ALL cases (Table 1) were enrolled in this study. The positive for JAK2-FGs were determined by G-banding karyotype analysis, transcriptome sequencing (RNA-seq; PMID: 34135310), reverse transcription PCR (RT-PCR), and interphase fluorescence in situ hybridization (I-FISH) with the JAK2 break-apart probe. Gene mutation analysis (PMID: 29932212) was also performed. According to whether there were FISH-JAK2 split signals in rod and lobed nuclear cells (Figure 1A), the cases were divided into My+ group (with myeloid involved) and My- group (without myeloid involved), respectively. Results Among these cases, there were 14 males and 7 females (M:F = 2:1); 15 B-ALL and 6 T-ALL cases; the median age was 22 years old (range 5-55). All cases were positive for JAK2-FGs in RNA-seq analysis, and all FGs were verified by RT-PCR and Sanger sequencing. A total of 16 JAK2 partner genes were detected (Table 1, Figure 1B/C), of which FNBP1, CDK5RAP2, GTF2I, and C14orf 93 have not been reported previously. All cases retained the complete kinase domain of the JAK2 gene, and 13 cases retained part or all of the JAK2 pseudo-kinase domain in their JAK2-FGs. Karyotype abnormalities were detected in 17 cases (81%), with 9p24 involved in 10 cases (48%), abnormalities other than 9p24 were observed in 7 cases, and karyotype was normal in 4 patients. FISH-JAK2 inspection showed split signals in 20 cases and showed negative results in case #21 who with a complex karyotype abnormality. Twelve cases (12/20=60%) showed JAK2 split signals in rotundum, rod, and lobed nuclei (Table 1, My+ group), suggesting both lymphoid and myeloid lineages were involved. We further investigated samples of 4 cases at the time of morphological complete remission after induction chemotherapy, of which the immunophenotype analysis was also negative for lymphoid blast cells. FISH-JAK2 inspection showed split signals accounting for 13%~79% of the nucleus, with split signals in the rod/lobed nucleus, and RT-PCR also confirmed abundant JAK2-FG transcripts in these samples. There were 8 B-ALL and 4 T-ALL cases in the My+ group, of which 2 T-ALL cases were also positive for STIL::TAL1 fusion, and 3 cases experienced eosinophilia during the course of the disease (Table 1). The 5 cases whose JAK2 partner genes were lymphoid development-related transcription factors (ETV6, PAX5) were all in the My- group. All My- cases and #21 had no documented eosinophilia. Among the 21 cases in this cohort, 13 had white blood cell (WBC) counts >50×10 9/L at diagnosis, and no significant hepatosplenomegaly. The total follow-up time was 2-89m, with a median overall survival (OS) time of 17m. Fourteen cases underwent allo-HSCT, and the median OS was 29.5m in the HSCT group and 9m in the non-HSCT group, respectively. The median OS was 15.5m in the My+ group and 29m in the My- group. The leukemia cells in these cases are prone to be in a high and rapid proliferation state, and they generally have a short OS and dismal prognosis. Conclusions Myeloid involvement accounted for 60% of JAK2-FG positive ALL and My+ cases had a worse prognosis and shorter OS than My- cases in this study. I-FISH inspection can be used as a method to determine whether there was myeloid involvement and provide valuable information for accurate analysis. The incidence of JAK2-FGs is low in ALL, but the fusion partners are diverse. This group of cases is more likely to have cryptic JAK2 translocations and with dismal prognosis. The phenotype of JAK2-FG malignancies is jointly determined by multiple factors under the influence of partner genes, accompanying fusion genes, and accompanying mutations.

Septin11 and Septin 8 Play Critical and Overlapping Roles Murine Stem Cell Function

Septins are highly conserved GTP binding proteins which regulate a variety of cellular functions including cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis and are now considered the fourth component of the cytoskeleton. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previous data in our laboratory led to the discovery of a small molecular inhibitor, DW0254, which inhibits RAS function and its downstream target RhoGTPase, Rac, including the hematopoietic-specific Rac2 GTPase, also implicated in MLL leukemias. The inhibitor leads to apoptosis of leukemia cell lines. Using biophysical methods, we discovered that DW0254 binds both the RAS chaperone, PDE6D, and Septin11, which is a member of the Septin6 family (Sara CN, et al., Blood Cancer 2022). Although Septins have been reported to be involved in function of the RhoGTPase Cdc42 and hematopoietic stem progenitor cell (HSPC) polarity, the role of Septin11 in hematopoiesis has not been reported. Septin11 is a member of the Septin6 family of proteins which consists of Septin6, 8, 10, 11 and 14 with significant homology between family members. Previous data in yeast has hypothesized functional overlap within each family of Septin proteins. To better understand the functional role of Septin11, we created lentivirus vectors tagged with a GFP reporter that contained shmiRs against Septin11. Culture of Septin11 knockdown (Septin11 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 13-fold decrease in cell counts compared to controls (p=0.02) and a decrease in total clonogenic activity (Figure 1) with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies. Reduced clonogenic numbers were accompanied by a significant increase in early apoptotic cells suggesting that Septin11 is important in regulating apoptosis in HSPC. To determine the potential role of Septin11 in more primitive hematopoietic cells, we utilized competitive repopulation assays. Transduced BoyJ HSPC (GFP+ Septin11 KD and BFP+ control, mixed 1:1) were injected into lethally irradiated C57Bl/6 mice. We observed a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary transplant recipients after knockdown of Septin11 (Table 1). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin11 most clearly in LT-HSC and ST-HSC (Table 1) with no statistically significant change in MPP. Therefore, to better study the hematopoietic stem cell compartment, we performed secondary transplants utilizing the cells obtained from primary competitive repopulation assays. We observed an unexpected recovery in engraftment in secondary transplant recipients that was accompanied by an increase in the mRNA expression of Septin8. Since Septin8 and Septin11 are closely related we hypothesized that induction of Septin8 was subserving key functions of Septin11. To test this hypothesis, we created a lentivirus vector that expressed both a shmiR against Septin11 and the Septin8 cDNA. Concurrent overexpression of Septin8 with Septin11 knockdown HSPC caused a significant increase in the total number of colonies compared to controls (Figure 1). These data indicate an important role of Septin11 in murine hematopoiesis and a potential redundant role for Septin8.

Transcriptomic without Clinical Response to Menin Inhibition As a Mechanism of Upfront Resistance in Samples from Mutliply Relapsed Patients with KMT2A-Rearranged Leukemia

Inhibitors of the Menin-KMT2A interaction are emerging as promising agents for the treatment of KMT2A-rearranged ( KMT2A-r) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Menin is required for the recruitment of the KMT2A-fusion to a subset of its target genes. Early phase clinical trials have shown highly encouraging results in patients with relapsed and refractory leukemias with KMT2A rearrangements and NPM1 mutations. In a recently reported early phase clinical trial evaluating the Menin inhibitor revumenib, about 50% of patients failed to show a clinical response despite robust downregulation of known Menin:KMT2A target genes, suggesting on target activity of the drug. This was the most common pattern of upfront resistance. We sought to understand upfront resistance (in contrast to the reported induced resistance via MEN1 mutations) in patient derived xenograft models. To this end, we evaluated the efficacy of Menin-inhibition in KMT2A-r leukemias with high-risk features: high-risk KMT2A fusion partners in AML ( MLLT10, MLLT4), infant ALL, and serial samples from patients with multiply relapsed ALL. We found that AML samples with high-risk fusion partners banked at initial diagnosis were sensitive to Menin-inhibition in xenografts. In contrast, three highly pretreated samples from patients with KMT2A-AFF1 ALL showed only a slight, or no decrease in latentcy in the Menin-inhibitor arm. Interestingly, Menin inhibition showed excellent in vivo efficacy against leukemia samples obtained from the same three patients earlier in their disease course. Therefore, prior chemotherapy (+/- immunotherapy) selects for Menin-inhibitor resistant cells without prior exposure to Menin inhibitor. Transcriptomic analysis (RNA-Seq) documents sustained on-target efficacy of the Menin inhibitor on a transcriptional level in resistant cells. None of the samples contained a MEN1 mutation at any time point. Targeted panel sequencing at initial diagnosis and relapse documented clonal evolution and the emergence of multiple co-mutations (particularly RAS pathway mutations and TP53 mutations), however, all co-mutations tested failed to induce resistance as a single alteration. It is possible that the cooperation of multiple emerging mutations (rather than a single mutation) is sufficient to drive AML in a KMT2A-fusion independent manner, thus mediating Menin resistance. A transcriptomic response (i.e. downregulation of KMT2A-fusion target genes) without a clinical response was the most common pattern of upfront resistance in the recently reported phase I/II clinical trial of the Menin inhibitor revumenib. We conclude that it will be critical to use Menin-inhibitors upfront or in early lines of therapy before substantial clonal evolution has occurred.

Ex Vivo Drug Sensitivity Evaluation of a ZMYM2::FGFR1 Fusion-Positive 8p11 Myeloproliferative Syndrome (EMS) Leukemia

Introduction : Fibroblast growth factor receptors (FGFRs) are conserved single transmembrane receptor tyrosine kinases (RTKs) with an extracellular ligand-binding domain and a cytoplasmic tyrosine kinase domain. They play vital roles in cellular proliferation, survival, development, fetal organogenesis, metabolism, and tissue repair. 8p11 myeloproliferative syndrome (EMS) is a hematologic malignancy caused by a translocation of the fibroblast growth factor receptor 1 (FGFR1) gene on chromosome 8p11-12. The most common fusion partner of FGFR1 is the zinc-finger domain ZNF198 (also known as ZMYM2) on chromosome 13q12. Pemigatinib is approved for FGFR fusion positive myeloid/lymphoid neoplasms (MLNs), and other FGFR inhibitors are being tested in clinical trials. However, EMS patients still face a poor prognosis and drug resistance, necessitating further research to overcome these challenges. This study evaluated ex vivo drug screening assay data generated from ZMYM2::FGFR1 positive primary leukemia samples throughout the course of an EMS patient's clinical response and validated these findings on a ZMYM2::FGFR1 positive cell line. Methods : Ex vivo and drug screening experiments were performed on freshly isolated primary patient samples and a ZMYM2::FGFR1-expressing cell line. The fusion was confirmed using FISH, and additional clinical specimens were collected with informed patient consent. Ex vivo drug sensitivity assays were conducted on patient mononuclear cells isolated from blood and bone marrow. IL-3 dependent BaF3 cells were infected with retrovirus containing a ZMYM2::FGFR1-V5 construct. ZMYM2::FGFR1 transformed BaF3 cells were evaluated with single agent and drug combination panels. Sanger sequencing of the FGFR portion of the fusion was performed on patient samples to confirm the breakpoint and identify potential mutations affecting sensitivity to Ponatinib throughout the course of treatment. Results : Sample were obtained with informed consent from a 36-year-old man diagnosed with 8p11 myeloproliferative syndrome and a ZMYM2::FGFR1 fusion along with a RUNX1 R201Q mutation. Ex vivo drug screens performed on isolated mononuclear cells demonstrated sensitivity to Ponatinib and Pemigatinib. He underwent treatment with HyperCVAD plus Ponatinib and subsequently HyperCVAD with Ponatinib and Velcade (Bortezomib) to CR1, FISH for FGFR1 rearrangement was undetected. He was scheduled to proceed with hematopoietic stem cell transplant (HSCT), however his pre-transplant bone marrow biopsy showed return of his FGFR1 translocation by FISH. Ex vivo drug screening assays confirmed resistance to Ponatinib, but continued sensitivity to Pemigatinib. He was transitioned to treatment with single agent Pemigatinib and was able to re-clear his FGFR1 fusion by FISH, and has since undergone hematopoietic stem cell transplantation. He remains in remission on post-transplant Pemigatinib maintenance therapy. Sanger sequencing performed at the time of progression on Ponatinib identified a nucleotide alteration resulting in an amino acid substitution (FGFR1 F686L; ZMYM2::FGFR1 F1171L) within the kinase domain that could contribute to decreased Ponatinib sensitivity. Bortezomib and Axitinib exhibited high efficacy on the patient's sample. Ex vivo drug assays performed on ZMYM2::FGFR1 transformed BaF3 cells confirmed the efficacy of Pemigatinib and Ponatinib. Other FGFR inhibitors, including Olverematinib, AZD4547, Axitinib, Cediranib, Dovitinib, and Lenvatinib, also demonstrated exquisite sensitivity. Despite extensive screening, no other single agents or drug combinations exhibited increased effectiveness in the ZMYM2::FGFR1 transformed BaF3 cells except for Trametinib, a MEK inhibitor, and the combination of Belvarafenib (RAF inhibitor) and Gilteritinib (FLT3 inhibitor). Conclusions : Ex vivo drug sensitivity assays demonstrated the highly selective efficacy of FGFR inhibitors in ZMYM2::FGFR1 fusion-positive leukemia cells and a fusion-expressing BaF3 cell line. Mutations in the FGFR1 kinase domain (ZMYM2::FGFR1 F1171L) could contribute to Ponatinib insensitivity. These ex vivo drug screening results provide further support for ongoing clinical trials which are investigating the use of single agent Pemigatinib and other FGFR1 inhibitors for the treatment of patients with FGFR1-fusion positive leukemias.

Integrated Methylation and Transcriptional Landscape and Evolution of Pediatric AML

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with poor outcomes despite intensive therapies. The genomic landscape of pediatric AML has been well characterized and has led to translational benefits, such as improved relapse predication and effective targeted therapies for a subset of individuals (e.g., FLT3-ITD AML). Despite these advances, the majority of patients continue to receive therapeutic interventions that have not changed in three decades. This underscores the need to advance our understanding of AML biology beyond genomic alterations (e.g., somatic mutations). Here, we present the largest (N = 858 patients) integrative analysis of pediatric AML paired transcriptomes and methylomes to date, performed at diagnosis for patients enrolled on Children's Oncology Group clinical trials AAML0531 (NCT00372593) or AAML1031 (NCT01371981). We also analyzed N = 400 and 253 corresponding transcriptomes and methylomes at relapse, respectively. Additionally, we performed comprehensive mutation and fusion calling from NGS data and integrated clinical covariates and outcomes. This robust dataset allowed us to uncover how pediatric AML epigenetics are perturbed from normal hematopoiesis, characterize the evolution of transcriptional and methylation changes from leukemia diagnosis to relapse, and identify more accurate relapse prediction models for pediatric AML. We initially performed unbiased hierarchical clustering on CpG sites that are differentially methylated between AML and normal bone marrow to identify distinct AML-restricted methylation (ARM) signatures. This analysis demonstrated that that these ARM clusters were enriched in specific cytomolecular subtypes of pediatric AML. Specifically, we identified methylation signatures that recapitulated their adult counterparts (e.g., IDH1/2, DNMT3A, WT1) and pediatric specific methylation signatures that clustered with RUNX1::RUNX1T1, CBFB::MYH11, KMT2A-rearrangement, NUP98::NSD1, NUP98::KDM5A, and CEBPA mutant AML. Next, we investigated the transcriptional and methylation signatures for the relapsed cases and compared them to diagnosis. We demonstrated that relapsed transcriptional and methylation signatures clustered within their cytomolecular subtype counterparts at diagnosis (i.e., AMLs at relapse more closely resemble AMLs at diagnosis with the same underlying gene fusion than other relapsed AMLs) ( Fig. 1A). However, analyzing our data in the context of these cytomolecular subtypes, we found that leukemias uniformly acquired a more stem-like state at relapse. We focused our biomarker discovery efforts on KMT2A-rearranged AMLs since (as a cohort) these patients have an intermediate risk of relapse and existing molecular biomarkers (e.g., KMT2A fusion partner and measurable residual disease detection) result in only modest improvements in relapse prediction. First, we found that CpG islands associated with CD34 and HOXA gene expression were frequently (but variably) hypermethylated, which was also associated with downregulation of CD34 and HOXA gene expression and favorable survival outcomes. Next, we performed non-negative matrix factorization on diagnosis and relapsed KMT2A-rearranged AMLs and identified a novel gene set associated with relapse and poor outcomes. These three distinct methylation-transcriptional findings (i.e., CD34, HOXA, and relapsed signatures) were subsequently transformed into a novel methylation-transcription biomarker score (MTBS) using machine learning algorithms (i.e., LASSO Cox regression). We used KMT2A-rearranged AML patients from AAML1031 as our training cohort (N = 129) to generate a MTBS that was able to distinguish KMT2A-rearranged cases with disparate outcomes. This algorithm was validated in an independent cohort from AAML0531 (N = 28) ( Fig. 1B). Additional methylation-transcriptional biomarker discovery efforts for pediatric AML are underway. Since pediatric AML is associated with a low mutational burden, understanding leukemia-specific transcriptional and epigenetic alterations is of utmost importance. Our study demonstrates the translational promise offered by deep functional genomic characterization of pediatric AML and we believe that further interrogation may provide additional novel insights into leukemia initiation, relapse prediction, and novel treatment approaches.

Asciminib Is Effective Against ABL1 Gene Fusions in Acute Lymphoblastic Leukemia but Only When the ABL1 SH3 Domain Is Present

Background Ph-like acute lymphoblastic leukemia (ALL) is high risk and comprised of many kinase activating gene fusions including those involving the ABL1 gene ( ABL1-rearranged, ABL1r). ABL1r ALL is associated with poor relapse-free and overall survival and incidence increases with age. Tyrosine kinase inhibitors (TKIs) such as dasatinib effectively target BCR::ABL1+ ALL and are in clinical trials for ABL1r ALL. However, resistance to TKIs is often observed, and novel treatments are required. The allosteric inhibitor asciminib binds the myristate pocket of Bcr-Abl kinase and is currently in Phase III trials for treatment of BCR::ABL1+leukemias. We assessed asciminib efficacy in ABL1r ALL. Methods Ba/F3 cells were transduced with four ABL1 fusions observed in ALL and asciminib efficacy evaluated in AnnexinV/7-AAD cell death assays (LD 50). Specific regions of the ABL1 fusion partner were deleted by site-directed mutagenesis (SDM). STAT5/CRKL phosphorylation and hCD45 levels were investigated by flow cytometry. Cells from three NUP214::ABL1 ALL patients were injected into NSG mice to establish patient-derived xenograft (PDX) models. Once mice reached 5% hCD45+ cells in peripheral blood (PB), treatment was commenced: vehicle control, dasatinib, asciminib. At experimental endpoint (>50% hCD45+ cells in PB) mice were humanely killed and organs harvested for analysis. Results Transduced Ba/F3 cells demonstrated varying sensitivity to asciminib (Table 1). While asciminib was not efficacious against Ba/F3 RCSD1::ABL1 and SNX2::ABL1 cells, Ba/F3 RANBP2::ABL1 (Heatley et al 2021)and NUP214::ABL1 cells demonstrated LD 50 concentrations within the clinically achievable range (3 µM, p<0.0001; 0.8 µM, p=0.0238 compared with control, respectively). Following asciminib treatment, Ba/F3 NUP214::ABL1 cells demonstrated reduced phosphorylation of STAT5 (mean fluorescence intensity [MFI]=1820 to 790, 57% reduction) and CRKL (MFI=2069 to 951, 54% reduction), confirming asciminib inhibits kinase signalling. No phosphorylation of STAT5 or CRKL was observed in control cells (MFI=290 and 406, STAT5 and CRKL, respectively). Additionally, ex vivo assessment of ETV6::ABL1 patient blasts demonstrated reduced phosphorylation of STAT5 (MFI=3395 to 1788, 47% reduction) and CRKL (MFI=5039 to 3000, 40% reduction) following asciminib treatment. Interrogation of the different ABL1 fusion breakpoints (Figure 1) revealed fusions demonstrating asciminib sensitivity retained exons 2 or 3 of ABL1 (corresponding to complete/partial SH3 and complete SH2 domains). Conversely, gene fusions lacking exons 1-3 (truncating the SH3 domain and retaining a partial SH2 domain) were resistant to asciminib suggesting a critical region in the SH3 and/or SH2 domain/s required for asciminib efficacy. To further investigate this we systematically deleted regions of ABL1 exon 3 using the NUP214::ABL1 construct as the model system and generated transduced Ba/F3 cells expressing different SDM regions. Cell death assays indicated the SH3 domain is critical for asciminib efficacy (Table 1). Asciminib also demonstrated efficacy in pre-clinical mouse modelsof NUP214::ABL1 ALL (where ABL1 exon 3 and partial SH3 domain is retained). Asciminib treatment significantly increased survival outcomes compared with control mice (84 vs 46d, p=0.01) by a time comparable to that of dasatinib treatment (93 vs 46d, p=0.0046). Results were confirmed in two additional NUP214::ABL1 PDX models demonstrating asciminib's potential to treat NUP214::ABL1 patients. Asciminib treatment reduced spleen and liver weights to that of healthy mice and resolved the leukemic immunophenotype of PB and bone marrow cells. Conclusion ABL1r ALL is associated with high rates of treatment failure and relapse. For the first time, we demonstrate the efficacy of asciminib in this high risk disease. Our data support addition of asciminib to treatment regimens of patients with NUP214::ABL1 ALL, the most common ABL class gene fusion affecting up to 6% of B-ALL patients and up to 10% of T-ALL patients. We also identify a critical region of ABL1 required for asciminib efficacy in ABL1r ALL. We propose interrogation of ABL1r patient breakpoints to determine inclusion/exclusion of the ABL1 SH3 domain as a surrogate test to be used in conjunction with in vitro kinase sensitivity assays for predicting whether a patient is likely to respond to asciminib treatment.

Purine Metabolism Modulates Leukemia Stem Cell Maintenance in MLL-Rearranged Acute Leukemia

Acute myeloid leukemia (AML) represents the most prevalent hematopoietic malignancy driven by leukemia stem cells (LSCs) and remains challenging to treat in adult patients. MLL rearrangement results in a genetically unique subtype of AML characterized by chromosomal translocations involving the MLL gene and over 80 fusion partners, including AF9 as one of the most common partners. The oncogenic MLL fusion proteins interact with multiple transcription and chromatin regulators, such as Menin, to induce leukemogenic stem cell gene programs and transform hematopoietic stem and progenitor cells. Although significant progress has been made in understanding the metabolic dysregulation in acute leukemia, such as alterations in glycolysis, energetic metabolism, amino acid metabolism, and fatty acid metabolism, the metabolic factors that modulate MLL fusion oncogenic transcriptional activity in AML remain incompletely elucidated. To identify novel metabolic vulnerabilities in MLL-rearranged AML, we performed a whole genome CRISPR dropout screen and profiled the metabolites of murine LSCs of MLL-AF9 murine model. This analysis revealed the purine biosynthesis pathway is essential for MLL-rearranged leukemia and that MLL-rearranged LSCs are enriched in metabolites of the purine biosynthesis pathway. Isotypoe incorporation experiments using [U- 13C]glucose demonstrated a high purine biosynthetic rate in the LSCs compared to bulk leukemia cells. Accordingly, human acute myeloid leukemia stem cells express high levels of the purine biosynthetic genes. Furthermore, we found that MYC, a well-known downstream target of MLL-AF9, mediates the enhanced purine synthesis in LSCs. These results demonstrate enhanced purine biosynthetic pathways in LSCs. We utilized genetic and pharmacological (mycophenolate mofetil, MMF, a purine biosynthesis inhibitor of IMPDH) approaches to target the enhanced purine metabolism and found inhibition of the purine synthesis pathway promotes myeloid differentiation of both murine and human LSCs. Metabolomics analysis reveals a remarkable reduction of purine metabolites. Supplementing the LSCs with guanosine, an intermediate metabolite of the purine biosynthesis pathway, substantially rescued the differentiation. Importantly, treatment of MLL-AF9-driven murine leukemia mice with MMF impaired LSCs function, prolonged leukemia survival, and reduced leukemia burden in the peripheral blood and bone marrow, while causing minimal effects on normal hematopoiesis. These results demonstrate the reliance on enhanced purine metabolism in LSCs maintenance. Previous work has shown that the purine biosynthesis pathway is essential for rRNA transcription in the nucleolus, which could be inhibited by CX-5461. To verify the essentiality of the rRNA transcription in MLL-rearranged LSCs, we treated MLL-AF9-induced murine LSCs cells with CX-5461 and found CX-5461 promotes myeloid differentiation of LSCs. Moreover, we found that the myeloid differentiation induced by MMF or CX-5461 is associated with reduced chromatin occupancy of the MLL-AF9 complex, especially Menin, and downregulated expression of MLL-AF9 target genes, such as Meis1, Hoxa9 and Myc, suggesting a regulatory role of nucleolar rRNA transcription on MLL-AF9 oncogenic gene expression program. We are currently focusing on the synergistic effects of targeting purine metabolism and Menin inhibition on AML suppression. Altogether, our findings reveal that purine metabolism maintains nucleolar rRNA transcription homeostasis to modulate MLL fusion complex-induced leukemogenic transcriptional activity in LSCs. The enhanced purine metabolism emerges as a crucial dependency for LSCs, providing potential targets for novel therapeutic strategies in treating MLL-rearranged leukemia.

Disease Monitoring in Acute Myeloid Leukemia with CLEC2A qRT-PCR

CLEC2A, C-Type Lectin Domain Family 2 Member A, is a newly discovered AML-restricted antigen. Detailed characteristics, clinical correlations, and therapeutic potential of CLEC2A in AML is presented by Kirkey et. Al. (ASH 2023). CLEC2A is entirely silent in normal hematopoiesis and uniquely expressed in patients with KMT2A rearrangements, providing an ideal therapeutic target in this high-risk subtype of AML. To date, CLEC2A has not been implicated in hematologic malignancies and expression of CLEC2A is entirely absent from normal tissues other than minimal expression in the skin. In addition to its utility as a therapeutic target, CLEC2A provides a unique target for disease monitoring given its lack of expression in normal hematopoietic cells. Here we present a highly sensitive quantitative molecular assay for CLEC2A, establishing its utility for disease monitoring in patients with KMT2A-r AML. In our efforts to evaluate CLEC2A expression and demonstrate the utility of using CLEC2A for disease monitoring in KMT2A-r, we developed a qRT-PCR assay using probes directed at exon 3 of the CLEC2A gene. The assay was optimized with RNA from the OCI-AML2 cells, validated to be CLEC2A-positive by flow cytometry and RNA from Kasumi-1 and K562 cells, both CLEC2A-negative by flow cytometry. We established the sensitivity of the assay through serial dilution of OCI-AML2 in a background of CLEC2A-negative Kasumi-1 or K562 cell RNA with concentrations ranging from 100% to 0.0001% OCI-AML2 RNA. The sensitivity assay demonstrated CLEC2A was detected at a level of 0.001% in both Kasumi-1 and K562. Given the association of CLEC2A with KMT2A-r AML, we evaluated the prevalence of CLEC2A as assessed by qRT-PCR. We screened diagnostic specimens from a large cohort of patients enrolled on recent COG Phase III clinical trials (ages 0-29) with and without KMT2A fusions to establish prevalence of CLEC2A expression. Using our CLEC2A qRT-PCR assay we evaluated diagnostic specimens from 122 patients with (N=76) and without (N=46) KMT2A-r, in addition to RNA from 8 normal bone marrow (NBM) samples, as controls and validated CLEC2A was entirely absent in NBM with median expression 0.13 copies per 1e 4 GUSB, figure 1A. Of evaluable samples, CLEC2A was detected in 65 of 76 (85%) KMT2A-r cases with a median expression of 317.45 copies per 1e 4 GUSB (range 0.03 - 21134.21 copies per 1e 4 GUSB). CLEC2A was detected in 21 of 46 (45%) non-KMT2A-r cases tested with a median expression of 1.37 copies per 1e 4 GUSB (range 0 - 1204.84 copies per 1e 4 GUSB). Further evaluation of KMT2A fusion partner showed that CLEC2A was detected in 100% of MLLT4 (N=6/6), 83.3% in MLLT10 (N=20/24), 72.7% of ELL (N=8/11), and 42.9% in MLLT3 (N=21/49). While CLEC2A expression was seen in both KMT2A-r and non-KMT2A-r AML patients, CLEC2A levels were significantly higher in KMT2A-r patients compared to non-KMT2A-r and NBM samples (p < 0.0001). Since CLEC2A is expressed in AML with no expression in normal hematopoietic cells, we hypothesized that monitoring CLEC2A via qRT-PCR throughout treatment (i.e., at diagnosis, at end of induction [EOI], at relapse) would provide added specificity to traditional flow cytometric disease monitoring. Using paired RNA samples from patients across multiple timepoints, we evaluated the change in CLEC2A expression throughout treatment, figure 1B. Patients with high CLEC2A expression at diagnosis (mean 1670 copies per 1e 4 GUSB, n=39) had decreased expression at EOI (mean 199 copies per 1e 4 GUSB, n=45) indicating treatment response, however high CLEC2A expression was seen at time of relapse (mean 1849 copies per 1e 4 GUSB, n=13) with CLEC2A expression preserved at relapse for all patients. qRT-PCR was able to detect CLEC2A expression in 7 patients at EOI who were in a morphologic CR with 6/7 (86%) patients being MRD-negative, further highlighting the increased sensitivity of CLEC2A qRT-PCR. Here we describe qRT-PCR monitoring of CLEC2A, a novel AML-restricted antigen highly enriched in KMT2A-r AML, provides added specificity and sensitivity to traditional flow cytometric MRD analysis. CLEC2A monitoring via qRT-PCR can be used to assess disease response throughout treatment and evaluate impending relapse as CLEC2A expression is retained at relapse. Additionally, qRT-PCR monitoring of CLEC2A can be incorporated in to pre- and post-HSCT disease evaluations similarly to qRT-PCR analysis of other known high-risk fusions such as NUP98-NSD1 fusions.

Comparative Atomistic Insights on Apo and ATP-I1171N/S/T in Nonsmall-Cell Lung Cancer.

Anaplastic lymphoma kinase (ALK) rearrangements occur in about 5% of nonsmall cell lung cancer (NSCLC) patients. Despite being first recognized as EML4-ALK, fusions with several additional genes have been identified, all of which cause constitutive activation of the ALK kinase and subsequently lead to tumor development. ALK inhibitors first-line crizotinib, second-line ceritinib, and alectinib are effective against NSCLC patients with these rearrangements. Patients progressing on crizotinib had various mutations in the ALK kinase domain. ALK fusion proteins are activated by oligomerization through the fusion partner, which leads to the autophosphorylation of the kinase's domain and consequent downstream activation. The proposed computational study focuses on understanding the activation mechanism of ALK and ATP binding of wild-type (WT) and I1171N/S/T mutations. We analyzed the conformational change of ALK I1171N/S/T mutations and ATP binding using molecular docking and molecular dynamics simulation approaches. According to principal component analysis and free energy landscape, it is clear that I1171N/S/T mutations in Apo and ATP showed different energy minima/unstable structures compared to WT-Apo. The results revealed that I1171N/S/T mutations and ATP binding significantly supported a change toward an active-state conformation, whereas WT-Apo remained inactive. We demonstrated that I1171N/S/T mutations are persistent in an active state and independent of ATP. The I1171S/T mutations showed greater intermolecular H-bonds with ATP than WT-ATP. The molecular mechanics Poisson-Boltzmann surface area analysis revealed that the I1171N/S/T mutation binding energy was similar to that of WT-ATP. This study shows that I1171N/S/T can form stable bonds with ATP and may contribute to a constitutively active kinase. Based on the Y1278-C1097 H-bond and E1167-K1150 salt bridge interaction, I1171N strongly promotes the constitutively active kinase independent of ATP. This structural mechanism study will aid in understanding the oncogenic activity of ALK and the basis for improving the ALK inhibitors.

Analysis of CD74 Occurrence in Oncogenic Fusion Proteins.

CD74 is a type II cell surface receptor found to be highly expressed in several hematological and solid cancers, due to its ability to activate pathways associated with tumor cell survival and proliferation. Over the past 16 years, CD74 has emerged as a commonly detected fusion partner in multiple oncogenic fusion proteins. Studies have found CD74 fusion proteins in a range of cancers, including lung adenocarcinoma, inflammatory breast cancer, and pediatric acute lymphoblastic leukemia. To date, there are five known CD74 fusion proteins, CD74-ROS1, CD74-NTRK1, CD74-NRG1, CD74-NRG2α, and CD74-PDGFRB, with a total of 16 different variants, each with unique genetic signatures. Importantly, the occurrence of CD74 in the formation of fusion proteins has not been well explored despite the fact that ROS1 and NRG1 families utilize CD74 as the primary partner for the formation of oncogenic fusions. Fusion proteins known to be oncogenic drivers, including those of CD74, are typically detected and targeted after standard chemotherapeutic plans fail and the disease relapses. The analysis reported herein provides insights into the early intervention of CD74 fusions and highlights the need for improved routine assessment methods so that targeted therapies can be applied while they are most effective.

Discovery, Structure-Activity Relationship and In Vitro Anticancer Activity of Small-Molecule Inhibitors of the Protein-Protein Interactions between AF9/ENL and AF4 or DOT1L.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause 5-10% acute leukemias with poor clinical outcomes. Protein-protein interactions (PPI) between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are drug targets for MLL-rearranged (MLL-r) leukemia. Several benzothiophene-carboxamide compounds were identified as novel inhibitors of these PPIs with IC50 values as low as 1.6 μM. Structure-activity relationship studies of 77 benzothiophene and related indole and benzofuran compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl substituent is essential for the activity. The inhibitors suppressed expression of MLL target genes HoxA9, Meis1 and Myc, and selectively inhibited proliferation of MLL-r and other acute myeloid leukemia cells with EC50 values as low as 4.7 μM. These inhibitors are useful chemical probes for biological studies of AF9/ENL, as well as pharmacological leads for further drug development against MLL-r and other leukemias.

AML with t(4;12)(q12;p13): A Detailed Genomic and Transcriptomic Analysis Reveals Genomic Breakpoint Heterogeneity, Absence of Pdgfra Fusion Transcripts and Presence of Pdgfra Overexpression in a Subset of Cases

Background: Gene fusions are a frequent event in acute myeloid leukaemia (AML). It is estimated that about 40% of AML patients harbour a cytogenetic abnormality resulting in a gene fusion. However, AML with t(4;12)(q12;p13) is very rare, but it is often accompanied by an aggressive clinical course and a poor prognosis. As frequently indicated by fluorescence in situ hybridization analyses (FISH), patients are assumed to harbor an ETV6::PDGFRA fusion, which impacts the choice of therapy, as these patients can be treated with tyrosine kinase inhibitors. The genomic region on chromosome 4 involved in ETV6 rearrangements is densly packed with genes in close spatial proximity. Therefore, the resolution of chromosome banding analysis and also FISH is not sufficient to fully elucidate the fusion partners involved. Recent studies have pointed out that many of these patients do not harbor an ETV6::PDGFRA fusion but other rather unknown fusion partners have been elucidated. Aim: (1) Evaluation of the genomic breakpoints in patients with t(4;12)(q12;p13) ETV6-rearranged AML; (2) determine the alteration of PDGFRA expression in patients with t(4;12). Patients and Methods: 10 patients (F: 4, M: 6; median age: 63 [44-82] years) with a t(4;12)(q12;p13) indicated by FISH and chromosome analysis were included. Samples were analysed by whole genome and whole transcriptome sequencing (WGS/WTS). Sequencing was performed on NovaSeq instrument (Illumina). Fusions were called with Arriba, STAR-Fusion and Manta. For gene expression analysis WTS data were log2 transformed and normalized with the trimmed mean of M-values (TMM) method. Results: Based on chromosome analysis and FISH patients showed break events in 4q12 and 12p13. In all cases a FISH signal constellation typical for an ETV6 rearrangement was observed. The mutation spectrum for each patient of the most commonly mutated genes in AML was examined by use of WGS data. The median number of mutations was 3, with mutations in ASXL1 being the most common (6 patients) and none of the patients featuring mutations in the PDGFRA gene. WTS analysis revealed in-frame fusions in 6 of 10 patients involving genes located on chromosomes 4 and 12 (4 patients with ETV6::CHIC2, 1 patients with ETV6::SCFD2, 1 patient with ETV6::ADAMTS3). None of the 10 examined patients showed a gene fusion, or a fusion transcript involving PDGFRA. Based on WTS data, 7 of the 10 patients showed a PDGFRA overexpression. WGS breakpoint analysis showed that genetic breakpoints on chromosome 12 cluster in introns 1 (4 patients) and 2 (6 patients) of the ETV6 gene. In contrast chromosome breaks on chromosome 4 are heterogeneously distributed in the 4q12 region. Breakpoint comparison indicated that patients with chromosome breaks in a range from chr4:54,804,230-54,962,206 (7 patients) display PDGFRA overexpression. In contrast patients with chromosome breaks further downstream chr4:55,021,511-73,409,362 (3 patients) did not exhibit an increased PDGFRA expression. This suggests an ectopic expression of PDGFRA triggered by the translocation and independent of the existence of an in-frame fusion transcript. A chromosome break in the range of chr4:54,804,230-54,962,206 (seen in 7 patients) might result in the inactivation of a CTCF binding site (chr4:54,796,345-54,796,360) upstream of these chromosome breaks that is already known to induce PDGFRA overexpression when disrupted. Conclusions: Analysis of AML with t(4;12)(q12;p13) translocation by WGS and WTS provides detailed information that separates two subsets distinguished by distinct breakpoint cluster regions on 4q12 and PDGFRA gene expression. The clinical impact of increased PDGFRA gene expression on response to TKI and prognosis as well as the presence of additional fusion transcripts has to be evaluated in further studies.

Deep and Durable Cytogenetic and Molecular Responses with Pemigatinib in Myeloid/Lymphoid Neoplasms with Fibroblast Growth Factor Receptor 1 Rearrangement: The Fight-203 Study

Introduction:Myeloid/lymphoid neoplasmswith fibroblast growth factor receptor 1 rearrangement (MLN FGFR1) are aggressive hematologic neoplasms caused by various reciprocal translocations involving chromosome band 8p11, resulting in tyrosine kinase fusion genes (eg, ZMYM2::FGFR1 from t[8;13][p11;q12] or BCR::FGFR1 from t[8;22][p11;q11]) and constitutive FGFR1 activationand downstream signaling. Pemigatinib is a potent and selective oral inhibitor of FGFR1-3 approved for the treatment of adults with relapsed or refractory MLN FGFR1. Efficacy and safety data for 41 patients with MLN FGFR1 in the ongoing FIGHT-203 study of pemigatinib were previously reported. Median daily pemigatinib dose was 9.5 mg; treatment-emergent adverse events led to dose interruptions in 66% of patients. Rates of complete response (CR) among the 38 patients evaluable for clinical response and rates of complete cytogenetic response (CCyR) for the 40 patients evaluable for cytogenetic response were 74% and 70%, respectively. CR and CCyR occurred in >80% of patients with chronic phase disease and in >50% of patients with blast phase disease. Here we present an analysis of centralized fluorescence in situ hybridization (FISH) evaluation of bone marrow (BM) cells and serial characterization of levels of FGFR1 fusion transcripts in this same cohort. Methods: FIGHT-203 (NCT03011372) is an ongoing open-label, multicenter, phase 2 study evaluating pemigatinib in patients ≥18 years old with MLN FGFR1 and ≥1 prior therapy. The starting dose was pemigatinib 13.5 mg daily (2 weeks on, 1 week off). Following protocol amendments, patients without prior therapy were eligible to enroll, and the dosing regimen was changed to a continuous schedule. Patients were censored for cytogenetic and molecular follow-up at the time of transplantation. CCyR was defined as 0 metaphases positive for the respective reciprocal translocation in ≥20 evaluated BM metaphases on local and/or central karyotyping or 0 cells (or not exceeding the lower level of detection of the probe) with the FGFR1 rearrangement in ≥200 BM cells on local and/or central break-apart FISH assays (central FISH prioritized). As part of the translational research plan, fusion partner genes were identified with next-generation sequencing (NGS; FusionPlex, Archer, Boulder, CO, USA) and targeted PCR profiling of RNA extracted from whole blood; droplet digital PCR (ddPCR) assays were developed to monitor the frequencies of specific FGFR1 fusion transcripts in RNA extracted from whole blood samples (estimated limit of detection, 0.05 copies/μL). Results: As of June 30, 2021, 39 of 41 enrolled and treated patients were evaluable for translational analyses. Among the 39 patients with whole blood samples, FGFR1 fusion partner genes identified on translational testing were ZMYM2 (n=17), BCR (n=11), FGFR1OP (n=4), TPR (n=3), TRIM24 (n=2), and C14orf93, CCDC6, CNTRL and ETV6 (n=1 each). Two of 39 patients had 2 fusions detected ( ZMYM2 and BCR, n=1; ZMYM2 and TPR, n=1). C14orf93, CCDC6 and ETV6 have not been previously identified as FGFR1 fusion partner genes. Central FISH assessment of BM cells confirmed FGFR1-aberrantcells were reduced to undetectable levels in responding patients but rebounded in some patients during periods of treatment interruption (Figure A). Serial ddPCR of whole blood samples demonstrated marked reductions in FGFR1 fusion transcripts for the 34 patients with ≥3 measures while on treatment (Figure B). Among patients with CR, 81.0% had a >2-log reduction and 47.6% had a >3-log reduction in detectable FGFR1 fusion transcripts. Smaller reductions in FGFR1 fusion transcripts were observed in patients with stable disease. Changes in FGFR1 fusion transcripts correlated with FGFR1 FISH results (Pearson coefficient = 0.81) and reflected changes in treatment intensity. Conclusion:Several new FGFR1 fusion partner genes, including C14orf93, CCDC6, and ETV6 were identified with NGS and PCR profiling. Pemigatinib treatment resulted in marked decreases in the percentages of cells with the FGFR1 rearrangement on FISH, as well as 2-3 log reductions in FGFR1 fusion transcripts in patients with MLN FGFR1. Further investigations are ongoing to determine the relationship between pemigatinib dose intensity and the depth and durability of molecular response.

Identification and Functional Validation of Neoantigen-Specific T Cells in Pediatric Patients with Fusion-Derived Acute Leukemias

Leukemia emerges from the accumulation of genetic mutations in hematopoietic cells, resulting in formation of altered proteins. Some of these mutant proteins (neoantigens) are consequently processed and presented as neoepitopes through major histocompatibility complex (MHC) molecules expressed on the surface of leukemia cells, permitting tumor-specific immune targeting. While adoptive T cell therapies against high mutation burden tumors have shown promising results, less is known about leveraging tumor-specific T cells as an effective approach in pediatric patients, including high-risk leukemia. In this study, peripheral blood or bone marrow samples were obtained from 34 leukemia patients aged 0 -14 years old. The patient tumors had founder fusion genes, including 15 patients with acute lymphoblastic leukemia (ALL), 15 patients with acute myeloid leukemia (AML), and 4 patients with mixed phenotype acute leukemia (MPAL). Samples were collected at the time of diagnosis or relapse. While leukemia blasts were the most abundant cellular component in these samples, the frequency of lymphocytes ranged between 1%- 25%, with a median of 3.5%. KMT2A rearrangements were expressed with either AFF1, MLLT1, MLLT3, MLLT4, MLLT6, or MLLT10 fusion partner in 73.5% of the patients' samples in this study. This included (14/15) ALL, (10/15) AML, and (1/4) MPAL patients . T cells were enriched and expanded for a total of 14 days using a rapid expansion protocol culture system. This approach was successful on all tested samples irrespective of their mutation backgrounds or initial lymphocyte count, with a range of 133-4957fold increases in total cell number and average frequencies of 59.7% and 26.1% for CD4+ and CD8+ T cell, respectively. We further screened the T cell response by examining the expression of 4-1BB, OX40, and IFN-g upon coculture with their autologous leukemia cells, and additionally validated the T cell killing activity against the blasts by a cell killing assay using standard protocol. We found that the expanded T cells exhibited a robust immune activation when cocultured with the autologous leukemia cells from ALL patients with KMT2A rearrangements, indicating an endogenous cell response in all patients in the ALL cohort (100%). However, we observed a considerably reduced reactivity in the samples from the AML and MPAL patients. Interestingly, we observed that such diminished reactivity was associated with a marked downregulation in the expression of MHC class I and II molecules, and that pretreating the leukemia blasts with IFN-γ prior to coculturing with the expanded T cells was sufficient to induce the expression of MHC molecules and subsequent activation of the expanded T cells. Single-cell TCR sequencing of the sorted, tumor-reactive T cells identified the major clonotypes among these reactive T cell clones and the top 4 - 6 clonotypes were selected from 5 patients to evaluate reactivity against the leukemia blasts and examine their specificity against the corresponding driving fusions. We further generated TCR-expressing clones and subsequently cocultured each clone with leukemia blasts to validate their antigen recognition and reactivity by cytotoxicity assay. Interestingly, and in line with the findings from investigations on solid tumors, the leukemia- reactive T cells were consistently characterized by higher expression of both PD1 and CD39 (PD1 hi CD39 +), indicating the capability of utilizing both markers for the enrichment of T cell prior to expansion and subsequent implementation. Despite the high-risk prognosis and low mutation burden in our patient cohort, our findings demonstrate that patient T cells contain endogenous neoantigen-specific clones which can be therapeutically leveraged for targeting leukemia blasts. These results support the utilization of adoptive T cell therapy in the context of hematological malignancies especially given the less restrictive spatial segregation between tumor-specific T cells and leukemia cells compared to solid tumors, where T cell infiltration is a prerequisite for antitumor immunity. Overall, these findings suggest the possibility of leveraging leukemia-associated lymphocytes, fusion oncogene vaccines, and TCR-based therapy as novel immunotherapeutic approaches for mitigating pediatric leukemia, particularly in high-risk patients.

Reclassification of ETS Family Transcription Factor Fusions in Pediatric AML Based on Molecular Drivers

Pediatric Acute Myeloid Leukemia (pAML) patients with a fusion involving an E26 transformation-specific (ETS) transcription factor have traditionally been considered high-risk. The most common fusions include ETV6::MNX1 t(7;12)(q36;p13) and FUS::ERG t(16;21)(p11;q22), yet primary fusion alone has proven incapable of predicting response to treatment. Indeed, within the 60 pAML patients with ETS fusions from the Children's Oncology Group (COG) trials AAML0531 and AAML1031, there were 25 unique fusions and diverse outcomes between patients who did share a primary fusion. A molecular based reclassification of ETS fused pAML could prove beneficial in the development of novel therapeutic additions to standard induction. Here, using genetic and RNAseq data from over 1,400 pediatric patients from AAML0531 and AAML1031, we reclassified ETS patients based on oncogenic transcriptional profiles as well as access to hematopoietic stem cell transplant (HSCT) ( Figure 1A). (19/60) patients presented with an enhanced MYC transcriptional signature (p-val < 0.00) independent of fusion partners [EP300::ETV6, ETV6::CCND3, ETV6::FAM133B, ETV6::FOXO1, ETV6::INO80D, ETV6::LMBR1, ETV6::MNX1, EWSR1::FEV, FLI1::IFIT2, FUS::ERG, FUS::FEV, FUS::FLI1]. Median event-free survival (EFS) was 293.5 days; median overall survival (OS) was 620.5 days. Notably, 100% (19/19) of patients with a MYC-driven signature relapsed; of the six patients who received HCST, five died within 3 years of diagnosis. Preliminary in vitro data in ETS cell lines with an enhanced MYC transcriptional signature (YNH-1, TSU-1621) suggests that the BET bromodomain inhibitor JQ1 may dampen MYC and induce anti-proliferative effects. (7/60) patients were defined by an EZH2-driven “immune-cold” signature characterized by global loss of immune receptors. Patients presented at diagnosis with significantly elevated EZH2 and low levels of MHC I and II receptors typically observed at relapse; additionally, there was significant downregulation in T cell and natural killer cell ligands and a 100% failure rate for HSCT. Median EFS was 255 days and median OS was 339 days. We have previously discussed the mechanism of leukemogenesis as well as proposed therapeutic interventions for this subset (Buteyn, ASH 2021). (34/60) patients did not possess either a MYC-driven or EZH2-driven signature. These “other” ETS were instead defined by whether or not the patient underwent HCST. (21/34) patients did not receive HCST and had median EFS and OS of 429 and 1194 days, respectively. The relapsed or refractory rate in this population was still 75%; (8/14) of R/R patients were deceased within three years. The remaining (13/60) ETS patients did receive HSCT in first remission. (11/13) are still alive and at a rate higher than the general pAML population post-HSCT (62.6%). Despite a survival rate at approximately 85%, ‘Other ETS +HSCT’ patients were all still categorized as ‘High Risk’ at the end of AAML1031. Overall, reclassification based on molecular drivers appears to not only be more accurate in predicting relapse and final outcome, but also in proposing novel agents for pAML patients who overwhelmingly do not respond to standard regimens. Reinvigoration of immune recognition by inhibition of EZH2 and/or inhibition of MYC-driven proliferation via BET inhibitors may prove an essential step in setting the stage for successful HSCT for a significant portion of pAML ETS patients.

A New t(7;9)(p12;q34) Involving NOTCH1 and IKZF1 in Pediatric T-Cell Lymphoblastic Lymphoma

Background Notch1 pathway activation is demonstrated in approximately 50% of T-cell acute lymphoblastic leukemias/lymphomas (T-ALL/LL) mainly by the presence of activating somatic mutations of NOTCH1 involving homodimerization and/or PEST (proline, glutamic acid, serine, threonine) domains. The oncogenic role of NOTCH1 was however primarily described with the characterization of the recurrent although rare t(7;9)(q34;q34). By positioning the coding region of the intracellular part of Notch1 (ICN1) under the control of the TCR beta-gene promoter, this translocation generates a truncated Notch1 receptor resulting in constitutive activation of the Notch1 pathway, independent of ligand binding. In murine models ICN1 is much more pro-oncogenic than NOTCH1 activating mutations. While several retrospective studies identified NOTCH1 activating mutations as an independent good-prognostic factor in human T-ALL/LL, search for truncated ICN1 in NOTCH1 wild-type patients was usually not performed/considered. Aim To describe and characterize a new translocation in a pediatric patient with T-LL. Method Somatic cytogenetics of blastic T-cells from diagnosis was explored by conventional karyotype. A specific fluorescence in situ hybridization (FISH) Breakapart probe was designed to explore the NOTCH1 locus on chromosome 9q34. Targeted locus amplification based sequencing (TLA) using genomic DNA was used to confirm/determine the partners involved in the new fusion found. Targeted RNA-sequencing using the Illumina TruSight RNA Fusion Panel was used to determine the putative transcript produced by the fusion. Results The patient, a 14-year-old young female, was diagnosed with stage 3 T-LL characterized by a large mediastinal mass with massive pleural effusion. Flow cytometry demonstrated a CD7+, CD1a+, CD4+/CD8+ double positive phenotype corresponding to EGIL type III T-LL. Conventional karyotype on T-lymphoblastic cells showed two clonal abnormalities, a deletion del(6)(q13q22) and a translocation t(7;9)(p12;q34) in 10 metaphases analyzed. Involvement of NOTCH1 in this new t(7;9)(p12;q34) was confirmed by molecular cytogenetics using a FISH Breakapart probe targeting the NOTCH1 locus. Targeted locus amplification based sequencing (TLA) revealed IKZF1 as the fusion partner involved in this new fusion. DNA sequencing revealed a predictive fusion between the intracellular domain of NOTCH1 (ICN1; breakpoint in intron 26 (NM_017617.5)) and IKF1 (breakpoint in intron 5 (NM_006060)). The targeted RNA-sequencing performed on the pleural effusion confirmed an in frame NOTCH1-IKZF1 fusion transcript. Sequencing of exons 26-27 and 34 of NOTCH1 showed wild-type alleles. After a swift response to the induction phase, the patient is in complete remission one year after completing her treatment according to the EuroLB-02 protocol. Summary/perspectives This new t(7;9)(p12;q34) involving ICN1 illustrates the importance to look for ICN1 more extensively in parallel to NOTCH1/FBXW7 mutations in the screening of Notch1 activation pathway in T-ALL/LL specially in the event of a stratification on Notch1 status for treatment. As both NOTCH1 and IKZF1 are major actors of normal T-cell differentiation/proliferation, the oncogenic role of the fusion NOTCH1::IKZF1 in T-ALL/LL deserves a better understanding in a larger population and will be further explored.

Combining Menin and MEK Inhibition to Target Poor Prognostic KMT2A-Rearranged RAS Pathway-Mutant Acute Leukemia

Approximately 10% of all acute leukemias harbor a chromosomal translocation involving the Mixed Lineage Leukemia 1 ( MLL1/ KMT2A) gene locus with over 90 fusion partners. These fusions are especially prevalent in pediatric acute leukemias. The resulting KMT2A fusion proteins (KMT2A-r) drive leukemogenic gene expression through an interaction with a chromatin complex that includes the scaffold protein menin, giving rise to aggressive acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In infant ALL, frequently characterized by KMT2A-r, the presence of RAS pathway mutations has been associated with a poor prognosis. The prognostic impact of RAS pathway mutations in pediatric KMT2A-r AML is less established. We thus analyzed data from 1605 pediatric patients with AML treated on the Children's Oncology Group (COG) AAML0531 and AAML1031 studies, with 336 of these having KMT2A-r. 51% (170/336) of these patients had mutations in NRAS, KRAS or PTPN11. The combination of KMT2A-r with RAS pathway mutations was associated with worse EFS and OS, HR=1.71 (p<0.001) for EFS and OS multivariable models. Given the prevalence of RAS pathway mutations co-occurring with KMTA-r, and the high-risk leukemia associated with this mutational profile, we sought to identify efficacious targeted drug combinations for this subset of childhood leukemia. Early clinical trial data for the menin inhibitor revumenib (SNDX-5613) demonstrated efficacy in KMT2A-r leukemias. Responding patients, however, typically develop progressive disease with a subset having mutations in MEN1 the gene encoding menin. Effective combination treatments for full efficacy of this targeted therapy are needed. We evaluated the combination of RAS/MAPK targeting using the MEK1/2 inhibitor selumetinib with VTP-50469, a close analog of revumenib, in pediatric leukemias harboring both KMT2A-r and RAS mutations. Treatment of AML and ALL cell lines and cells from an AML patient-derived xenograft (PDX) model in vitro with this drug combination resulted in a synergistic decrease in viability, a G0/G1 cell cycle arrest, and an increase in apoptosis. The combination also resulted in decreased AML colony formation. We next explored potential mechanisms by which the combination treatment had a synergistic anti-leukemic effect. Both selumetinib and VTP-50469 treatment individually reduced phosphorylated ERK levels, but the combination had the most complete inhibitory effect. The combination treatment also resulted in loss of stem-like gene expression signatures, an increase in myeloid differentiation programs, and most strikingly, downregulation of targets of MYC signaling. Both MYC transcript and protein levels were reduced by each drug alone and even more effectively with the combination. Next, we evaluated the in vivo efficacy of this drug combination in three orthotopic pediatric AML and ALL pediatric PDX models harboring different KMT2A-r and RAS mutations (AML: KMT2A- AF10 with KRAS, KMT2A- PICALM with NRAS, ALL: MLL- AF4 with NRAS). NSG mice engrafted with PDX models via tail vein injection were assigned to four treatment groups: vehicle, chow containing revumenib (0.03%), selumetinib (50 mg/kg/day, 5 days a week), or the combination. After treatment for 18-28 days leukemia burden in bone marrow, spleen and peripheral blood was markedly decreased with the combination compared to the single drug-treated cohorts in 2 models with RAS mutations. In one of the AML models, single agent treatment with revumenib or selumetinib was highly efficacious at eradicating disease at the measured time point. We also found a striking decrease in levels of MYC, P-ERK and MEIS1 by western immunoblotting, highlighting on-target activity of the drugs and supporting our in vitro data for a role for MYC in the synergistic effect observed with the combination. In conclusion, new clinical data from a large cohort of COG patients demonstrates that KMT2A-r and co-occurring RAS pathway mutations portend a distinctively worse outcome for children with AML. The findings in our preclinical study suggest a promising, readily translatable targeted treatment for this patient population. Mechanistically, enhanced downregulation of both MYC signaling and the RAS/MAPK pathway with the combinatorial therapy is likely a strong contributor to the observed efficacy.

New eGFP Mutant with Intact C- and N-Termini and Affinity for Ni<sup>2+</sup>

The green fluorescent protein GFP has long been used in research practice as a molecular tool. It is often used as a fusion partner. To create fusion constructs, target molecules are attached to the N- or C-terminus of GFP. On the other hand, the N- or C-termini of GFP required to create fusion constructs are also used to attach affinity tags that is greatly facilitating purification. Simultaneous introduction of affinity tag and GFP to both or the same end of GFP can create steric hindrances both in the process of biosynthetic folding of the construct and in its affinity purification. This work is devoted to the production of GFP with a His-tag introduced into the polypeptide chain. This work resulted in eGFP157_7H protein with an embedded His-tag and free N- and C-termini to create fusion proteins. The added His-tag will allow purification of the construct with GFP by metal-chelated affinity chromatography under native conditions. The resulting eGFP157_7H variant retained the original fluorescent properties completely similar to those of wild-type eGFP.