MLL Translocations Research Articles

Abstract 3330: BPP, a dual functional inhibitor, targets MLL-rearranged acute myeloid leukemia: Unraveling mechanisms and expanding therapeutic horizons

Abstract Refractory/recurrent acute myeloid leukemia (AML) poses a formidable clinical challenge, with MLL translocations implicated in approximately 10% of AML cases and a staggering 75% in infant leukemia, bearing an unfavorable prognosis. Our investigation focuses on BPP, a dual-functional inhibitor capable of inducing DNA cross-linking and suppressing angiogenesis. In a diverse panel of solid tumor and leukemic cells, BPP exhibited exceptional sensitivity against MLL-rearranged AML cell types. In MOLM13 and MV4-11 xenograft models, BPP showcased outstanding tumor suppression, with some tumors even achieving complete remission. In-house acute toxicity testing, BPP treated group showed excellent safety margin in both blood chemistry and pathological examination. Remarkably, BPP demonstrated significant cytotoxicity not only in FLT3-WT AML but also in FLT3-ITD AML. Delving into the mechanism, BPP was found to induce FLT3-ITD and MLL degradation by triggering DNA damage and ER stress. Comet assays revealed a dose-dependent accumulation of DNA fragments in MLL cells upon BPP exposure.The involvement of the KMT2A gene in chromosomal translocation in THP1 cells was highlighted, with BPP treatment resulting in a significant increase in Heat-shock protein. Notably, the combination of ganetespib, an FDA-approved HSP90 inhibitor, with BPP exhibited synergistic effects in both in vitro and in vivo models, inducing synthetic lethality and inhibiting HR repair. Furthermore, BPP not only reduced leukemic stem cells in colony formation assays but also demonstrated sensitivity in relapsed/refractory (R/R) and multiple drug-resistant patient samples. These findings collectively position BPP as a promising candidate for the treatment of MLL-rearranged AML, offering multifaceted therapeutic potential. Citation Format: Tai-Lin Chen, Tsann-Long Su, Te-Chang Lee. BPP, a dual functional inhibitor, targets MLL-rearranged acute myeloid leukemia: Unraveling mechanisms and expanding therapeutic horizons [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3330.

MC180295 is a highly potent and selective CDK9 inhibitor with preclinical in vitro and in vivo efficacy in cancer

BackgroundInhibition of cyclin-dependent kinase 9 (CDK9), a novel epigenetic target in cancer, can reactivate epigenetically silenced genes in cancer by dephosphorylating the SWI/SNF chromatin remodeler BRG1. Here, we characterized the anti-tumor efficacy of MC180295, a newly developed CDK9 inhibitor.MethodsIn this study, we explored the pharmacokinetics of MC180295 in mice and rats, and tested the anti-tumor efficacy of MC180295, and its enantiomers, in multiple cancer cell lines and mouse models. We also combined CDK9 inhibition with a DNA methyltransferase (DNMT) inhibitor, decitabine, in multiple mouse models, and tested MC180295 dependence on T cells. Drug toxicity was measured by checking body weights and complete blood counts.ResultsMC180295 had high specificity for CDK9 and high potency against multiple neoplastic cell lines (median IC50 of 171 nM in 46 cell lines representing 6 different malignancies), with the highest potency seen in AML cell lines derived from patients with MLL translocations. MC180295 is a racemic mixture of two enantiomers, MC180379 and MC180380, with MC180380 showing higher potency in a live-cell epigenetic assay. Both MC180295 and MC180380 showed efficacy in in vivo AML and colon cancer xenograft models, and significant synergy with decitabine in both cancer models. Lastly, we found that CDK9 inhibition-mediated anti-tumoral effects were partially dependent on CD8 + T cells in vivo, indicating a significant immune component to the response.ConclusionsMC180380, an inhibitor of cyclin-dependent kinase 9 (CDK9), is an efficacious anti-cancer agent worth advancing further toward clinical use.

Targeting EYA1 Activity in MLL- Rearranged Leukemia

The Mixed Lineage Leukemia gene (also known as MLL or KMT2A) is involved in chromosomal translocations in a subtype of acute myeloid (AML) and lymphoblastic leukemias (ALL). MLL translocations have been identified with more than 80 partner genes, and the most common AML translocation partner is MLLT3 (AF9). MLL-rearranged (MLL-r) leukemia is associated with a sudden onset, aggressive progression, and poor prognosis in comparison to non-MLL-r (MLL-nr) leukemias, and there is a need for more effective therapies. Previous work determined that AF9 directly interacts with multiple different regulatory proteins, including the BCL6 corepressor (BCOR). An AF9 point mutation (E531R) was identified that selectively disrupts BCOR binding to AF9 and MLL-AF9. The loss of BCOR binding to MLL-AF9 abrogated its leukemogenic ability in a mouse transplant model. RNA-seq analysis determined that of the MLL-AF9 direct target genes, EYA1 was the most down-regulated in the MLL-AF9 (E531R) point mutant-transformed cells. Addback of EYA1 into MLL-AF9 (E531R) cells partially rescued its leukemogenic ability, suggesting that EYA1 has an important role in MLL-AF9 leukemogenesis. EYA1 is a transcriptional coactivator and protein tyrosine phosphatase that is expressed during embryonic development and its re-expression has been implicated in several cancers, including MLL-r leukemia. The objective of this study is to investigate the in vitroand in vivo effects of EYA1 inhibition in MLL-AF9 transformed murine bone marrow stem/progenitor cells and in human MLL-rearranged (MLL-r) and non-rearranged (MLL-nr) leukemia cell lines. Cells were treated with small molecule inhibitors at varied concentrations and time points. Effects of inhibition were assessed with viability, cell morphology, cell cycle, surface and intracellular marker expression analyses, and in vivo transplant studies. EYA1 inhibition reduced cell viability by 50% - 90% in MLL-AF9 transformed murine bone marrow (mBM) cells and multiple human AML cell lines. EYA1 inhibition did not affect some human B-ALL cell lines, which also express undetectable levels of EYA1, indicating a correlation between the response to EYA1 inhibition and EYA1 expression. In MLL-r and MLL-nr cells, cell cycle analysis indicated a reduction in cells entering S-phase, stalling in G0/G1, with EYA1 inhibition as well as an induction in cellular senescence. Inhibiting EYA1 activity induced cell morphology changes toward a more differentiated state in MLL-AF9 mBM and several MLL-r and -nr cell lines. To confirm these qualitative results, cells were stained for myeloid differentiation markers, and cell surface expression of CD11b, Gr1, and CD14 increased in a dose- and time-dependent manner in all cell types, while HSC markers CD117 (c-KIT) and CD34 were reduced in MLL-AF9 mBM and human AML cells, respectively. In assessing the mechanism of EYA1 in MLL-r leukemia, MLL-AF9 cells treated with EYA1 inhibitors showed increased RNA pol II CTD Tyr1 phosphorylation levels (known EYA1 substrate) compared to vehicle-treated cells. Thus, unregulated EYA1 phosphatase activity targeting RNA Pol II CTD may contribute to leukemia development. Mice that received MLL-AF9 mBM cells treated with an EYA1 inhibitor had a significantly decreased rate of disease progression and increased survival compared to control. Bone marrow cells isolated from these mice showed increased expression of myeloid differentiation markers CD11b and Gr1 compared to vehicle-treated mice, confirming that EYA1 activity contributes significantly to leukemogenesis. The inhibition of EYA1 induces leukemia cell differentiation, cell cycle arrest and senescence, and cell death. Ongoing experiments include additional in vivo murine leukemia, further mechanistic analyses, and combinatorial experiments to evaluate the efficacy of a multi-target approach with EYA1 inhibition and other targeted-therapies. This work may provide a promising, novel therapeutic approach to MLL-r and MLL-nr leukemias that will help improve patient outcomes.

CRISPR-ChIP reveals selective regulation of H3K79me2 by Menin in MLL leukemia.

Chromatin regulation involves the selective recruitment of chromatin factors to facilitate DNA repair, replication and transcription. Here we demonstrate the utility of coupling unbiased functional genomics with chromatin immunoprecipitation (CRISPR-ChIP) to identify the factors associated with active chromatin modifications in mammalian cells. Specifically, an integrated reporter containing a cis-regulatory element of interest and a single guide RNA provide a chromatinized template for a direct readout for regulators of histone modifications associated with actively transcribed genes such as H3K4me3 and H3K79me2. With CRISPR-ChIP, we identify all the nonredundant COMPASS complex members required for H3K4me3 and demonstrate that RNA polymerase II is dispensable for the maintenance of H3K4me3. As H3K79me2 has a putative oncogenic function in leukemia cells driven by MLL translocations, using CRISPR-ChIP we reveal a functional partitioning of H3K79 methylation into two distinct regulatory units: an oncogenic DOT1L complex directed by the MLL fusion protein in a Menin-dependent manner and a separate endogenous DOT1L complex, where catalytic activity is directed by MLLT10. Overall, CRISPR-ChIP provides a powerful tool for the unbiased interrogation of the mechanisms underpinning chromatin regulation.

Abstract IA016: Characterization of the protein-protein interactions involved in hDOT1L epigenetic regulation on biochemical, structural and functional level towards developing new therapeutic intervention

Abstract MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. More than 90 different MLL translocation partners have been identified with the most common fusion partners, AF9, ENL, AF4, and AF10. AF9 and ENL are closely related members of the YEATS domain protein family and have been identified as part of several reported complexes including Super Elongation Complex (SEC) and DotCom Complex. AF9 and ENL, through their C-terminal hydrophobic domain, known as ANC1 Homology Domain (AHD), recruit the histone H3K79 methyltransferase hDOT1L to the MLL target genes, constitutively activating transcription of pro-leukemic targets. We have characterized the protein−protein interactions between hDOT1L and MLL fusion oncogenic proteins, AF9 and ENL, on the biochemical, biophysical, and functional level. This study mapped a 10 amino-acid region (aa 865− 874), highly conserved in hDOT1L from a variety of species, as an interacting site with the AHD domain of AF9/ENL proteins and demonstrated that these PPIs are essential for cellular immortalization mediated by MLL-AF9. Applying genetic approach and studying the effects of blocking the AF9-hDOT1L PPIs or hDOT1L enzymatic activity, it was demonstrated complete disruption of hDOT1L recruitment to critical target genes, Hoxa9 and Meis1, in cells expressing hDOT1L mutants with an impaired AF9 binding site. This was followed by significant decrease in H3K79me2 in the promoter regions of these genes, cell differentiation and induction of apoptosis. To identify the potential therapeutic benefits of this novel targeting approach, the effect of these different hDOT1L mutants was investigated on adult murine hematopoiesis. The in vivo studies demonstrated that genetic interventions that result in loss of hDOT1L enzymatic activity rapidly depletes hematopoietic stem and progenitor cells. In contrast, blocking the AF9-hDOT1L PPIs allowed for sustained hematopoiesis. These studies provided an important proof of concept for the potential therapeutic advantage of inhibiting the AF9-hDOT1L interaction and disrupting the integrity of the MLL-fusion complexes. In addition, these results demonstrated that hDOT1L recruitment can be abolished by a single point mutant (I867A), highlighting the crucial role of the hydrophobic interactions and giving insights for further drug discovery. We have reported the first peptidomimetics targeting PPIs between MLL-AF9 and hDOT1L, providing evidence for the druggability assessment of this novel potential therapeutic approach. The AF9/ENL C-terminal domain, is an intrinsically disordered protein, representing a challenge for structural studies. Applying X-ray crystallography, we have successfully determined the 3D structure of the AF9 AHD domain in complex with peptidomimetics establishing a platform for structure-based design of novel, optimized, potent inhibitors, the current focus of our research efforts. Citation Format: Zaneta Nikolovska-Coleska. Characterization of the protein-protein interactions involved in hDOT1L epigenetic regulation on biochemical, structural and functional level towards developing new therapeutic intervention. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA016.

The ENL YEATS epigenetic reader domain critically links MLL-ENL to leukemic stem cell frequency in t(11;19) Leukemia.

MLL (KMT2a) translocations are found in ~10% of acute leukemia patients, giving rise to oncogenic MLL-fusion proteins. A common MLL translocation partner is ENL and associated with a poor prognosis in t(11;19) patients. ENL contains a highly conserved N-terminal YEATS domain that binds acetylated histones and interacts with the PAF1c, an epigenetic regulator protein complex essential for MLL-fusion leukemogenesis. Recently, wild-type ENL, and specifically the YEATS domain, was shown to be essential for leukemic cell growth. However, the inclusion and importance of the YEATS domain in MLL-ENL-mediated leukemogenesis remains unexplored. We found the YEATS domain is retained in 84.1% of MLL-ENL patients and crucial for MLL-ENL-mediated leukemogenesis in mouse models. Mechanistically, deletion of the YEATS domain impaired MLL-ENL fusion protein binding and decreased expression of pro-leukemic genes like Eya1 and Meis1. Point mutations that disrupt YEATS domain binding to acetylated histones decreased stem cell frequency and increased MLL-ENL-mediated leukemia latency. Therapeutically, YEATS containing MLL-ENL leukemic cells display increased sensitivity to the YEATS inhibitor SGC-iMLLT compared to control AML cells. Our results demonstrate that the YEATS domain is important for MLL-ENL fusion protein-mediated leukemogenesis and exposes an "Achilles heel" that may be therapeutically targeted for treating t(11;19) patients.

Hoxa11-mediated reduction of cell migration contributes to myeloid sarcoma formation induced by cooperation of MLL/AF10 with activating KRAS mutation in a mouse transplantation model

The molecular mechanism of myeloid sarcoma (MS) formation remains nuclear. Our clinical and mouse model findings from a previous study revealed that cooperation of KMT2A (MLL) translocation (MLL-t) with activating N-/K-RAS mutations promoted MS formation in a shorter latency. To improve the understanding of MS formation, in this study, we performed imaging cell trafficking analysis and demonstrated that cells harboring cooperating mutations migrated more slowly to omental adipose tissues and more cells were retained in adipose tissues in vivo. Comparison of transcriptome profiling among three pairs of mouse MLL/AF10(OM-LZ) leukemia cell lines harboring activating and wild-type KRAS identified 77 differentially expressed genes (DEGs) with >1.5-fold change. Functional annotation of these 77 DEGs using Gene Ontology (GO) enrichment analysis followed by cluster analysis revealed that GO terms related to development/differentiation have the highest enrichment score. The roles of Hoxa10 and Hoxa11, two genes which mapped to this cluster, were further characterized. Silencing Hoxa10 and Hoxa11 in cells harboring cooperating mutations prolonged the survival and reduced MS formation, respectively, in the recipient mice. Data of imaging cell trafficking as well as competitive engraftment and clonal expansion analyses indicated that silencing or overexpressing Hoxa11 in mouse leukemia cells affected cell migration and retention in omental adipose tissue. Although silencing Hoxa11 in leukemia cells did not affect Cxcr4 expression, it resulted in increased transwell migration, motility in confined spaces 3 μm in size, and cell protrusion. Our results revealed that Hoxa10 plays an important role in survival and Hoxa11 contributes to MS formation in MLL-t acute myeloid leukemia with activating KRAS mutation.

ALL-258: Loss of Heterozygosity in STR Loci Found in Tumor DNA of De Nονο-Diagnosed ALL Patients as a Factor Predicting Poor Outcomes for B-ALL but Not T-ALL Patients

Context: Loss of heterozygosity (LOH) in some STR loci established for de novo-diagnosed ALL could be used as a prognostic factor for possible poor clinical outcomes. Objective: To identify LOH in the blast cells of patients with B- and T-ALL at diagnosis and to analyze therapy outcomes according to LOH status. Design: This study includes an analysis of the STR profiles in the DNA of tumor cells for a cohort of 87 patients with de novo-diagnosed Ph-negative ALL (40 B-ALL, 44 T-ALL, 3 biphenotypic ALL) and a statistical evaluation of LOH as a possible risk factor for B- and T-ALL. All patients were treated according to the “RALL-2016” regimen at the National Research Center for Hematology (Moscow, Russia). Setting: DNA was isolated from patient bone marrow samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission and/or from a buccal swab. STR profiles were assessed by PCR with the COrDIS Plus multiplex kit (Gordiz Ltd, Russia). LOH in tumor DNA was verified by chromosomal microarray (CMA CytoScanHD). Patients: Inclusion criteria: de novo-diagnosed Ph-negative ALL patients, 18-55 years old, intermediate-risk group without MLL translocation t(4;11)(q21;q23), and treatment by RALL-2016 protocol. Exclusion criteria: patients older than 55 years, MLL translocation t(4;11)(q21;q23), and pretreatment. Main Outcome Measures: A multivariate survival analysis was used to assess the independent impact of the LOH as a risk factor. Overall survival (OS) was chosen as the primary endpoint. Results: Of the 87 patients, 21 were characterized with LOH in certain STR loci (24%). The most common was 9p LOH (12 patients). 12p LOH was detected in 8 patients. According to OS analysis, LOH 12p seems to be a marker of a favorable prognosis. For B-ALL patients, LOH (excepting 12p LOH) was an independent risk factor (OS hazard ratio 3.89, log-rank P-value 0.0395). For T-ALL patients, the OS hazard ratio was 0.59 (log-rank P-value 0.62). Conclusions: We have found a statistically significant association of clinical failures with the LOH in STR loci measured at the onset of B-ALL but not T-ALL. Funding: This study was supported by Rakfond grant 5/2019

Discovery of M-1121 as an Orally Active Covalent Inhibitor of Menin-MLL Interaction Capable of Achieving Complete and Long-Lasting Tumor Regression.

Targeting the menin-MLL protein-protein interaction is being pursued as a new therapeutic strategy for the treatment of acute leukemia carrying MLL-rearrangements (MLLr leukemia). Herein, we report M-1121, a covalent and orally active inhibitor of the menin-MLL interaction capable of achieving complete and persistent tumor regression. M-1121 establishes covalent interactions with Cysteine 329 located in the MLL binding pocket of menin and potently inhibits growth of acute leukemia cell lines carrying MLL translocations with no activity in cell lines with wild-type MLL. Consistent with the mechanism of action, M-1121 drives dose-dependent down-regulation of HOXA9 and MEIS1 gene expression in the MLL-rearranged MV4;11 leukemia cell line. M-1121 is orally bioavailable and shows potent antitumor activity in vivo with tumor regressions observed at tolerated doses in the MV4;11 subcutaneous and disseminated models of MLL-rearranged leukemia. Together, our findings support development of an orally active covalent menin inhibitor as a new therapy for MLLr leukemia.

PHF6 Mutations Defines a Subgroup of Mixed Phenotype of Acute Leukemia with Aberrant T-Cell Differentiation

Mixed phenotype acute leukemia (MPAL) is a rare form of acute leukemia, and comprises 2% of all acute leukemia diagnoses. With the exception of a small subset harboring BCR/ABL1 or MLL translocations (around 15% in combination), the genetic aberrations of MPAL are largely unknown. PHF6 gene is located at Xq26-27 and encodes plant homeodomain finger 6, a zinc finger containing protein. Recurrent somatic PHF6 mutations are frequent in T-ALL (up to 30%) and rarely in AML (~3%). Here we report recurrent PHF6 mutations in approximately 20% of MPAL patients, making PHF6 the most common genetic aberrations identified in MPAL.From 2013 to 2016, a total of 1329 patients with myeloid neoplasms were sequenced at the MSKCC with a targeted next generation sequencing panel containing 30 genes that are frequently mutated in acute leukemia. We identified 38 PHF6 mutations with predicted pathologic significance in 33 patients (2.5%) spanning the whole coding sequence: 10 frameshift, 11 nonsense, 1 splicing site, and 16 missense (Figure 1). The most common co-mutant gene observed in cases with PHF6 mutations was RUNX1 (30%, 11/37). No TP53 mutations are detected within the PHF6-mutant cohort(Figure 2). The 33 patients with PHF6 mutations included patients with a diagnosis of MPAL (n=5), AML (n=19), MDS (n=5), and MPN (n=4).The disproportional distribution of PHF6 mutations in MPAL prompted us to further analyze these patients. During the same period of time, 24 MPAL patients were identified and confirmed based on WHO 2016 classification (excluding AML-MRC and t-AML). Within this cohort, 3 (12%) had BCR-ABL1 (p190) fusion, and 1 (4%) had MLL translocation, two distinct genetically defined subgroups. Importantly, 5 (21%) harbored PHF6 mutations, these mutations were mutually exclusive with BCR-ABL1 or MLL. Four of the 5 patients showed T-cell lineage differentiation, in contrast to MPAL with BCR-ABL1 or MLL translocations which were characterized by B/myeloid differentiation. The 2-year overall survival for the PHF6 mutation positive MPAL was markedly reduced compared to non-mutated cases (40% vs. 100% in PHF6 unmutated cases (p=0.005)).In the larger subset of AML cases, PHF6 mutations were nearly exclusively present in secondary AML (11 AML-MRC, 6 t-AML and only 1 de novo AML). In contrast to their rarity in de novo AML, PHF6 mutations were identified in 11% (11/88) of AML-MRC and 7.1% (6/84) of t-AML. Interestingly, T-cell markers (cytoplasmic CD3, CD2, CD5, CD7) were often expressed on the blasts of secondary AML cases regardless of PHF6 mutational status (56%, 9/16 vs. unmutated 51%, 36/70). In comparison to unmutated cases, the blasts were often negative for CD33 (69%, 11/16, vs 28%, 20/70, p=0.004 by Fisher's exact test) and CD38 (44%, 7/16, vs. 17%, 12/70, p=0.04 by Fisher's exact test) in PHF6 mutated AML, suggestive of a stemness phenotype with aberrant T-cell differentiation.In conclusion, PHF6 mutations are the most common somatic alteration identified in MPAL and define a subgroup of MPAL with altered T-cell differentiation and inferior outcome. Amongst AML, PHF6 mutations are almost exclusively seen in secondary AML and are also associated with T-cell antigen expression and a more primitive stem/progenitor immunophenotype. PHF6 mutations co-exist with RUNX1 mutations but are mutually exclusive to TP53 mutations. Notably, PHF6 mutations may provide a genetic and functional link between MPALs with poor outcome and secondary AML and provide insights into shared mechanisms of transformation. RNA sequencing studies on the sorted blast population are ongoing and will be presented at the ASH meeting. [Display omitted] DisclosuresArcila:Invivoscribe: Honoraria; Archer: Honoraria; Raindance Tecnologies: Honoraria. Levine:Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Inotuzumab Ozogamicin (InO) for Relapsed/Refractory Acute Lymphoblastic Leukemia (R/R ALL) in the Global Phase 3 INO-VATE Trial: Efficacy By MLL Status

Inotuzumab Ozogamicin (InO) for Relapsed/Refractory Acute Lymphoblastic Leukemia (R/R ALL) in the Global Phase 3 INO-VATE Trial: Efficacy By MLL Status

DNA Demethylating Agents Generate a Brcaness Effect in Multiple Sporadic Tumor Types: Prediction for Sensitivity to PARP Inhibitors in AML

Poly (ADP-ribose) polymerase inhibitors (PARPis) have shown clinical efficacy in breast and ovarian cancers with inherited mutations in BRCA genes that abrogate homologous recombination (HR) DNA double-strand break (DSB) repair activity. However, to date, PARPishave not had success in sporadic breast and ovarian cancers, or other cancers, such as acute myeloid leukemia (AML), with intact BRCA genes. We have recently reported that triple-negative breast cancer (TNBC) and AML are sensitive to low doses of the potent PARPiTalazoparib in combination with DNA methyl transferase inhibitors (DNMTi), decitabine (DAC) or azacitidine (AZA), in vitro and in vivo. In these cancer models, the DNMTi/PARPicombination increases amplitude and retention of PARP1 directly at laser-induced DNA damage sites, increasing DNA DSBs, promoting synergistic tumor cytotoxicity, blunting cell self-renewal, and leading to strong anti-tumor responses. These results have led to a Phase I/II Clinical trial of DNMTi/PARPi combination therapy in patients with relapsed/refractory AML that is underway.In identifying the molecular pathways that may underlie efficacy of this inhibitor combination in sporadic TNBC, ovarian cancer and AML, we find that low doses of DNMTisreprogram the cancer genome, in part by changing gene expression patterns of DSB repair genes, potentially creating a HR defect (HRD) or “BRCAness” effect. Expression array (Agilent Human 44K v2) analysis was performed on RNA extracted from multiple BRCA-intact sporadic cell lines from AML (including, Kasumi-1, KG-1 and MOLM-14), TNBC (including, MDA-MB-231, SUM159) and ovarian cancer (including, A2780) on days 1, 3, 5 and 7 and 10 post DNMTi(DAC 10nM or AZA 500nM) treatment. Hallmark, KEGG and reactome gene ontologies were utilized. Only gene sets with log fold change (FDR) >.250 were considered significant and genes demonstrating 2-fold or greater change with drug treatment were selected. While multiple DNA repair pathway genes were found both up- and down- regulated post DNMTi treatment, one or more HR genes, were consistently downregulated in all cell lines examined. QPCR of mRNA and/or immunoblotting of HR proteins, in particular RAD51, BRCA1, BRCA2, or HR-related genes, FEN1 or FANCD2, showed significantly decreased expression with DNMTi treatment (p<0.05) in TNBC, ovarian and AML cell lines, validating array data. Functional assays in TNBC cell lines, using a stably expressed HR repair reporter (DRGFP) also confirmed decreased HR activity with DNMTi treatment. Additionally, in response to DNA damage with ionizing radiation (IR, 4Gy), RAD51 foci were reduced in AZA-treated TNBC and ovarian cancer cell lines, compared to untreated controls. Significantly increased triradial chromosomes were also observed in TNBC in the presence of AZA when exposed to DNA cross-linking agents mitomycin C (MMC, 50ng/ml) or cisplatin (2μM) treatment, confirming functional down-regulation of the HR-related FANCD2 pathway.The above HRD-like or BRCAness phenotype with DNMTi treatment translated to sensitivity to PARPis in primary bone marrow mononuclear cells (BMMNC) from AML patients. Thus, in 6 of 8 (75%) AML samples, HR gene expression by QPCR of RNA and HR functional activity by immunostaining for RAD51 foci, following DAC treatment (10nM, 72 hrs), DNMTi treatment resulted in a significant decrease in HR or HR-related gene expression, including RAD51, BRCA1, BRCA2, and decreased levels of RAD51 foci (p<0.05). All 6 AML samples were also significantly more sensitive to DAC/talazoparib combination treatment (p<0.05) in colony forming assays, compared with single agent treatments. AML samples included those with MLL translocations that are reported to have high HR activity and resistance to PARPis. Studies are underway to elucidate the molecular characteristics of the minority of AML patient samples (N=2) that did not generate a BRCAness effect with DAC treatment. In conclusion, our data suggest that one of the mechanisms underlying DNMTi/PARPi therapy efficacy in AML is DNMTi-induced BRCAness. In vitro testing for BRCAness with DNMTitreatment could be a biomarker to predict sensitivity to DNMTi/PARPis and inform selection of AML patients for future clinical trials of this inhibitor combination. DisclosuresCivin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; 3DBioWork Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kingsbury:3DBioWorks: Consultancy, Equity Ownership.

Fetal and Neonatal Hematopoietic Progenitors Are Exquisitely Sensitive to MLL-ENL-Driven Leukemia Initiation

Translocations involving the MLL1/KMT2A gene are observed in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), but the frequency at which these mutations occurs is different at different stages of life. For example, MLL rearrangements are extremely common in infant leukemia (~70% of cases), but they are much less common in adolescent or adult AML (~5% of de novo adult AML). While the function of MLL fusion proteins has been extensively studied, it remains unclear why MLL-rearrangements are so common in infant leukemias as compared to adolescent or adult leukemias. One possibility is that fetal hematopoietic progenitors are more readily transformed by MLL fusion proteins than adult progenitors. Recent work in our lab has shown that a mutation commonly associated with adult AML, the FLT3-Internal Tandem Duplication, selectively activates leukemogenic transcriptional programs in adult as compared to fetal/neonatal progenitors (eLife 5:e18882). We have developed a system to test whether an analogous effect holds for MLL translocations - i.e. do they preferentially initiate leukemogenesis in fetal progenitors. We made use of a previously described tet-O doxycycline inducible mouse allele (Cell Reports 9:1246) to both spatially and temporally control expression of MLL-ENL, a common MLL fusion product (Fig. 1A). We found that MLL-ENL induction at fetal (E10.5) or neonatal (P0) stages caused AML to develop in most mice, but MLL-ENL induction at juvenile (P21) or adult (8 weeks) stages did not (Fig. 1B). Remarkably, AML developed with the greatest penetrance (100%) when MLL-ENL was induced at P0 as compared to both earlier and later developmental stages. In neonates, MLL-ENL profoundly depleted hematopoietic stem cells (HSCs) and, to a lesser extent, lineage-restricted hematopoietic progenitors (HPCs), and it caused expansion of myeloid progenitor populations. These effects began to dissipate by 10 weeks after birth. Changes in HSC and HPC frequency were not at all evident when MLL-ENL was induced at an adult stage, but by a year after MLL-ENL induction, the surviving mice had a significantly expanded HSC pool. MLL-ENL induced widespread changes in gene expression in neonatal HPCs. In contrast, very few changes in gene expression were observed in MLL-ENL expressing adult HPCs. Known targets of MLL-ENL, such as HoxA9, were more highly induced in neonatal HPCs than in adult HPCs, though expression was observed in older mice. Altogether, our data demonstrate a dynamic relationship between developmental stage and the transcriptional response to MLL-ENL, and they may help account for the high percentage of MLL rearrangements observed in infant leukemia. Furthermore, our findings have opened new lines of investigation into the relationship between the epigenetic landscapes of developing HSCs/HPCs and the programs that drive leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Genome-Wide, In Vivo, Dropout CRISPR Screen in Acute Myeloid Leukemia Identifies an Essential Role for Beta-Galactosylation in Leukemic Cell Homing

Genetic drivers of acute myeloid leukemia (AML) include chromosomal translocations and somatic mutations involving regulators of epigenetic, transcriptional, and cell-signaling pathways. Many of these mutations cannot be directly targeted with pharmacological agents. To circumvent the problem, a better functional characterization of the leukemic phenotype would provide additional therapeutic options. Murine in vivo models of AML, developed by introducing human AML mutations in murine hematopoietic progenitors, can recapitulate many features of the disease in a reproducible way. By coupling these models of AML with perturbations of selected genes, we can rapidly validate hypotheses regarding the growth requirements of leukemic cells in vivo. Specifically, these experiments can identify essential genes for AML even when these genes are not mutated. Hence, an experimental understanding of AML is complementary to the sequencing of human mutations.Pooled genetic perturbation screens performed in experimental models of malignancies are an efficient approach to identify genes essential to malignant cells. However, most screens reported so far have been performed in vitro, where cell growth conditions differ significantly from the malignant milieu in vivo. Here, we report the development of experimental and statistical tools that allowed us to identify positive and negative regulators of AML growth on a genomic scale in vivo. We created Cas9-expressing murine models of AML (mAML), amenable to genetic manipulation, by overexpressing the human AML (hAML) oncogenes HOXA9 and MEIS1, or the fusion protein MLL/MLLT3, in murine myeloid progenitors isolated from a Rosa26-Cas9-knockin mouse. While MLL translocations account for approximately 3-10% of AML cases, HOXA9 and MEIS1 are overexpressed in 70% of cases. Thus, these mAML tools collectively modeled a significant fraction of hAML. Cells were transduced ex vivo with genome-wide sgRNA libraries containing 130,209 sequences then transplanted in cohorts of recipients. The depletion in vivo of sgRNAs was compared to non-targeting library controls in the reconstituted leukemia to assess statistical significance. This gave us a genome-wide functional annotation of the leukemic genome in vivo. To validate the effect of the top 1034 genes identified in the screen, we created a validation library and tested its effect on leukemic cells and normal hematopoietic progenitors to identify lead candidates.Several genetic pathways showed greater levels of depletion in vivo compared to the in vitro arm of the screen. Among these was oxidative phosphorylation, suggesting that AML cells rely on it to a greater extent in vivo. We then focused on single genes which disruption in vivo had a much larger effect. As expected, we identified several immune regulators, partners in integrin-mediated adhesion and, interestingly, two regulators of beta-galactosylation, galactose epimerase (Gale) and beta-1,4-galactosyltransferase 1 (B4galt1). Genetically ablating this pathway, using CRISPR/Cas9, in AML cells had a statistically significant effect on survival of mice. Engraftment of AML cells was markedly reduced. Interestingly, the growth rates of leukemic or normal hematopoietic progenitors in vitro was not different between knock-out and control. Beta-galactosylation is known to post-translationally modify several proteins involved in homing and cell adhesion. Thus, we propose that altering beta-galactosylation in AML cells is a novel strategy to modulate their interaction with the bone marrow microenvironment for therapeutic benefit, which could be complementary to current approaches. DisclosuresNo relevant conflicts of interest to declare.

CCG 1953: Acute Lymphoblastic Leukemia in Infants: Analysis of Prognostic Factors: A Report from the Children's Oncology Group.

Background: Infant ALL has a poor therapeutic outcome. In contrast to other subtypes of ALL, infants have not benefited from risk-adapted therapy and intensification of therapy. This is the first cooperative group trial characterizing all infants at the molecular level for MLL/11q23 rearrangements for better stratification in future trials.Procedure: All infants enrolled on CCG 1953 were tested for MLL rearrangement by Southern Blot, and the 11q23 translocation partner was identified (4;11, 9;11, 11;19, or “other”) by reverse-transcriptase PCR.Results: 115 infants were enrolled (July 1996 to August 2000). Treatment for patients consisted of 4 courses of induction-consolidation-intensification followed by 3 cycles of intensified maintenance and 4 cycles of routine maintenance. Infants with 11q23/MLL abnormalities and a protocol-defined 5-6/6 related or unrelated donor identified within 4 months were assigned to BMT. Central nervous system prophylaxis consisted of triple intrathecal therapy during the first 10 weeks of therapy. Infants not undergoing BMT received additional CNS prophylaxis with very high dose methotrexate. Patients then received one year of intensified maintenance, followed by a year of standard maintenance. Overall event-free survival (EFS) was 41.7% (SD = 9.2%) and overall survival (OS) was 44.8% at 5 years. Five-year EFS for MLL rearranged cases was 33.6%, and for MLL non-rearranged cases was 60.3% (p=.006). 5-year EFS for MLL/11q23 subgroups was as follows: t(4;11): 29.0%; t(11;19): 30.0%; t(9;11): 22.2%; and “other 11q23” 53.5% (p=.122). Multivariate Cox regression analyses showed a rank order of significance for negative impact on prognosis for CD10 negativity, age less than 6 months, and MLL rearrangement, in that order. We did not observe a statistically significant difference in outcome between different MLL translocation partners, though we did observe a non-significant trend toward improved outcome in patients with “other” MLL translocation partners as compared to those with t(4;11), t(9;11) or t(11;19). Toxicity was the most frequent cause of death. Relapse as a first event in 1953 was later (median 295 days) compared to the predecessor1883 infant ALL trial (median 207 days).Conclusions: MLL/11q23 rearrangement, CD10 expression, and age are important prognostic factors in infant ALL, but individual molecular 11q23 translocation partners do not predict outcome. These results indicate that certain subgroups have an unusually poor outcome and are therefore candidates for novel therapeutic approaches in first remission. Given the high mortality, further dose intensification strategies are unlikely to result in better long-term survival.

Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors.

The MLL gene located at 11q23 is frequently disrupted by chromosomal translocation in a wide spectrum of newly diagnosed acute leukemias. Recently, it has become apparent that the MLL gene is very frequently disrupted by chromosomal translocations in patients with secondary leukemias associated with chemotherapeutic regimens incorporating topoisomerase II inhibitors. These secondary leukemias associated with topoisomerase II inhibitors (most commonly teniposide, etoposide, or doxorubicin) have distinct clinical and biologic features which have led to the speculation that they are induced by treatment with topoisomerase II inhibitors. We have identified a site within the MLL breakpoint cluster region (bcr) that is highly sensitive to double-strand DNA cleavage induced by topoisomerase II inhibitors. This finding is quite specific and highly reproducible. Although it was initially discovered in malignant lymphoblasts isolated from a patient receiving multiagent chemotherapy, this site-specific double-strand DNA cleavage can be induced in tissue culture using malignant cell lines as well as peripheral blood from normal individuals. Site-specific cleavage occurs in a significant fraction of cells using a variety of model systems, is both time and dose dependent, and can be induced with either doxorubicin or etoposide. This site-specific cleavage maps to the same region as a consensus topoisomerase II cleavage site within the MLL bcr. These results suggest that site specific cleavage within the MLL bcr induced by topoisomerase II inhibitors may be an early step leading to MLL translocations and secondary leukemia.

ALL-332: Loss of Heterozygosity in the Short Tandem Repeat (STR) Loci Found in Tum or DNA of De Novo-Diagnosed ALL Patients as a Factor Predicting Poor Outcome

Context Loss of heterozygosity (LOH) was described for many malignancies including leukemia. Previously, we noted LOH at STR loci is especially frequent for relapsed ALL patients. LOH established for de novo-diagnosed ALL could be used as a prognostic factor for possible poor clinical outcome. Objective To identify LOH in the blast cells of the patients with ALL at diagnosis and to analyze therapy outcomes relative to patients without LOH. Design This study includes an analysis of the STR profiles of the DNA of tumor cells from a cohort of 47 patients with de novo-diagnosed Ph-negative ALL undergoing treatment according to the “RALL-2016” regimen at the National Research Center for Hematology (Moscow, Russia) and statistical evaluation of LOH as a possible risk factor. Patients with the LOH at the short arm of 12 chromosome (12pLOH) are excluded from risk group because 12p LOH is not associated with worse outcome according to many publications. Setting DNA was isolated from patient bone marrow samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission and/or from the buccal swab. STR profiles were assessed by PCR with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). Patients Inclusion criteria: de novo-diagnosed Ph-negative ALL patients, 18–55 years old, intermediate risk group without MLL translocation t(4;11)(q21;q23), and treatment by RALL-2016 protocol. Exclusion criteria: patients older than 55 years, MLL translocation t(4;11)(q21;q23), and pre-treatment. Main outcome measures A multivariate survival analysis was used to assess the independent impact of the LOH as a risk factor. Overall survival (OS) was chosen as primary endpoint. Results Of the 47 patients, 10 were found with LOH in the certain STR loci (21%). 12p LOH was detected in four patients. OS of patients with other LOH was significantly lower than for patients without LOH (HR=6.02 (95% CI: 1.66–21.78), p=0.0019). Conclusions We have found a statistically significant association of clinical failures with the LOH in STR loci (except 12p LOH) measured at the onset of ALL. Funding This study was supported by Rakfond grant 5/2019

A Dysregulated DNA Methylation Landscape Linked to Gene Expression in MLL-Rearranged AML

ABSTRACT Translocations of the KMT2A (MLL) gene define a biologically distinct and clinically aggressive subtype of acute myeloid leukaemia (AML), marked by a characteristic gene expression profile and few cooperating mutations. Although dysregulation of the epigenetic landscape in this leukaemia is particularly interesting given the low mutation frequency, its comprehensive analysis using whole genome bisulphite sequencing (WGBS) has not been previously performed. Here we investigated epigenetic dysregulation in nine MLL-rearranged (MLL-r) AML samples by comparing them to six normal myeloid controls, using a computational method that encapsulates mean DNA methylation measurements along with analyses of methylation stochasticity. We discovered a dramatically altered epigenetic profile in MLL-r AML, associated with genome-wide hypomethylation and a markedly increased DNA methylation entropy reflecting an increasingly disordered epigenome. Methylation discordance mapped to key genes and regulatory elements that included bivalent promoters and active enhancers. Genes associated with significant changes in methylation stochasticity recapitulated known MLL-r AML expression signatures, suggesting a role for the altered epigenetic landscape in the transcriptional programme initiated by MLL translocations. Accordingly, we established statistically significant associations between discordances in methylation stochasticity and gene expression in MLL-r AML, thus providing a link between the altered epigenetic landscape and the phenotype.

MLL-Rearranged AML Is Associated with Poor Outcomes As Compared to Patients with Intermediate- and Adverse-Risk Disease: A CIBMTR Study of 3779 Adult Patients

Background Studies evaluating the impact of 11q23 / KMT2A / MLL translocations on post-transplant outcomes as compared to other intermediate- and adverse-risk cytogenetic characteristics are lacking. Methods We considered 8709 AML patients from the CIBMTR database for inclusion. We evaluated relapse, non-relapse mortality (NRM), disease-free survival (DFS), and overall survival (OS) for patients who were transplanted in CR1 stratified by presence of 11q23 rearrangement, intermediate- or adverse-risk disease. Assignment of cytogenetic risk was performed by hand, based on the 2010 AML-MRC Classification System. A multivariate analysis was performed using a Cox proportional hazards model. Results Characteristics of 3779 patients, selected based on disease and donor type, HSCT in CR1, and availability of data, are shown in Figure 1. Of these, 426 patients had an 11q23-rearrangement, 2384 had intermediate-risk disease, and 969 had adverse-risk disease. Of the patients with 11q23 translocations, 112 (26%) had t(9;11), 62 (15%) had t(11;19), 41 (10%) had t(6;11), 28 (7%) had t(10;11), and 47 (11%) had other partners. No partner was provided in 136 cases (32%). Figures 2 and 3 show the DFS, OS, NRM, and relapse data stratified by cytogenetic category. Based on the multivariate analysis, DFS was lowest in adverse-risk patients (HR for death 1.47, 1.26 for MLL, p Stratification of outcomes by translocation partner did not yield any statistically significant differences between groups, however a descriptive analysis of these data will be presented. Measurable residual disease (MRD) status did not appear to play an independent role in NRM or in OS. However, the multivariate HR for relapse in patients with MRD-positivity was 1.23, p Conclusions Patients with MLL-rearranged AML had post-HSCT DFS and OS that were more similar to patients with adverse-risk disease than to those with intermediate-risk characteristics. MLL patients had a higher probability of relapse than intermediate-, but not adverse-risk, patients. NRM was similar among all groups. Both prognostic classification and MRD status independently contributed to the risk of relapse and DFS, but MRD was not an independent factor in predicting NRM or OS. Our study suggests that patients with MLL rearrangements do almost as poorly as those with adverse-risk disease and that novel approaches for this high-risk population represent an urgent unmet clinical need.