MLL-r Leukemia Research Articles

Abstract C095: The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia

Abstract Introduction: Over 70% of infants with acute lymphoblastic leukemia (ALL) present with mixed lineage leukemia (MLL1/KMT2A) gene rearrangements (MLL-r ALL), which is associated with poor prognosis. MLL-r encodes oncogenic fusion proteins that cause aberrant chromatin histone methylation and leukemic transformation of hematopoietic progenitors. A characteristic of MLL-r leukemia is high expression of the FLT3 gene. Menin is critical for leukemic transformation by MLL fusion proteins. VTP-50469, the preclinical precursor of revumenib (SNDX-5613), is a potent and selective small molecule inhibitor of the Menin-MLL interaction that has shown impressive single-agent activity in preclinical models of MLL-r acute leukemia. Revumenib is currently undergoing clinical evaluation, therefore it was of interest to assess the in vivo activity of VTP-50469 in combination with established drugs against MLL-r ALL patient-derived xenografts (PDXs). Methods: VTP-50469 was tested as a single agent against a panel of 7 pediatric MLL-r ALL PDXs in immune-deficient (NSG) mice (120 mg/kg twice daily x 28 days via oral gavage). Combination studies used VTP-50469 formulated in chow (0.1%) available ad libitum for 28 days in combination with either an induction-type regimen of vincristine (0.15 mg/kg once weekly x 4 weeks), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off x 4 weeks, VXL) all administered intraperitoneally, or the FLT3 inhibitor gilteritinib (Xospata, 30 mg/kg x 28 days via oral gavage). Events were defined as >25% huCD45+ in the peripheral blood. Drug efficacy was assessed by event-free survival of treated (T) and control (C) groups by T-C, T/C measurements and stringent objective response criteria (progressive disease, PD; complete response, CR; maintained CR, MCR), and disease infiltration in the spleen and femoral bone marrow (BM) at 28 days post-treatment initiation. Results: Single-agent VTP-50469 elicited MCRs in 6/7 PDXs and significantly (P<0.0001) reduced leukemia infiltration of the spleen (7/7 PDXs) and BM (6/7 PDXs) compared to vehicle control mice. When administered in chow, VTP-50469 elicited a CR in the MLL-7 PDX (T-C 48.2 days, T/C 8.1) and a PD in MLL-8 (T-C 23.0 days, T/C 4.9). VTP-50469 combined with VXL elicited MCRs in both PDXs (MLL-7 T-C 77.0 days, T/C 12.3; MLL-8 T-C 49.1 days, T/C 9.3) and significantly delayed leukemia progression compared with VTP-50469 or VXL alone (P=0.0013). Moreover, VTP-50469 combined with VXL almost completely eradicated leukemia infiltration of spleens and BMs by both PDXs. VTP-50469 combined with gilteritinib did not result in therapeutic enhancement compared to each single agent arm in either PDX. Conclusions: The addition of VTP-50469 to a standard-of-care induction-type regimen (VXL) resulted in therapeutic enhancement against two infant MLL-r ALL PDXs, while VTP-50469 plus gilteritinib was substantially less effective. These results support further clinical evaluation of revumenib in combination with standard-of-care therapy in MLL-r leukemia. Citation Format: Richard B Lock, Kathryn Evans, Sophia Gawan-Taylor, Ben Watts, Timothy Stearns, Eric J Earley, Emily L Jocoy, Carol J Bult, Beverly A Teicher, Gerard M McGeehan, Malcolm A Smith. The menin inhibitor VTP-50469 enhances the in vivo efficacy of established drugs against preclinical models of aggressive infant MLL-r acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C095.

Systematic characterization of the HOXA9 downstream targets in MLL-r leukemia by noncoding CRISPR screens

Accumulating evidence indicates that HOXA9 dysregulation is necessary and sufficient for leukemic transformation and maintenance. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes. Here, we conduct dropout CRISPR screens against 229 HOXA9-bound peaks identified by ChIP-seq. Integrative data analysis identifies reproducible noncoding hits, including those located in the distal enhancer of FLT3 and intron of CDK6. The Cas9-editing and dCas9-KRAB silencing of the HOXA9-bound sites significantly reduce corresponding gene transcription and impair cell proliferation in vitro, and in vivo by transplantation into NSG female mice. In addition, RNA-seq, Q-PCR analysis, chromatin accessibility change, and chromatin conformation evaluation uncover the noncoding regulation mechanism of HOXA9 and its functional downstream genes. In summary, our work improves our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency.

BOD1L Facilitates Chromatin Binding of SETD1A and Promotes Leukemia Cell Growth

The H3K4 methyltransferase SETD1A plays an essential role in leukemia through a non-catalytic activity which regulates transcriptional pause release of RNA polymerase II. SETD1A localizes to the transcriptional start site of numerous actively transcribed genes, but primarily regulates the expression of a restricted set of genes within the DNA damage response as well as heme biosynthesis pathway. However, the roles of upstream regulators or essential components of SETD1A chromatin binding remain unclear. Using the DepMap database and a CRISPR-tiling screen method, we found that BOD1L is the most correlated co-dependency with SETD1A in human cancer cell lines and that the Shg1 domain of BOD1L plays an indispensable role in human MOLM-13 leukemia cell line. BOD1L knockout increases apoptosis and reduces cell cycle. The strong BOD1L dependency in AML cells was also confirmed using a mouse MLL-r leukemia model in vitro and in vivo. SETD1A catalytic domain mutant AML cells still responded to the BOD1L sgRNAs that suggests the role of BOD1L is independent from the SETD1A catalytic function. Importantly, BOD1L knockout mimics the transcriptional profiles of SETD1A knockout cells and reduces the expression of genes in DNA damage response and heme biosynthesis. BOD1L loss immediately reduces SETD1A distribution on chromatin and increased the NELFE and RNA polymerase II signals at transcriptional start sites. These results show that BOD1L is an essential component of the non-catalytic SETD1A function in transcriptional pause release. We also performed an immunoprecipitation assay using tagged constructs to examine the interaction between BOD1L and SETD1A. Our data indicate that SETD1A associates with BOD1L through the FLOS domain, especially in the C-terminal region. In addition, we found that the loop structure in the Shg1 domain of BOD1L is associated with the nonenzymatic domain of SETD1A. A split luciferase complementation assay reveals that the BOD1L Shg1 domain and SETD1A FLOS domain interact. To demonstrate the role of BOD1L chromatin interaction, we enforced localization of the Shg1 domain fragment at a silent gene locus and it successfully induced ectopic SETD1A recruitment to chromatin and formation of the COMPASS complex. However, we observed neither induced H3K4me3 modification nor RNA polymerase II recruitment. Taken together, while the BOD1L-SETD1A complex is not sufficient to act as a pioneer complex for active transcription, our study identified that BOD1L facilitates chromatin binding of SETD1A through the Shg1-FLOS domain interaction and then regulates transcriptional pause release, and that this interaction is a potential therapeutic opportunity for SETD1A-targeting therapy.

MLL (KMT2A)-rearranged Acute Lymphoblastic Leukemias Are Addicted to S-Adenosyl Methionine (SAM): Implications for Therapy

MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is an aggressive subset associated with poor survival and relapse representing 5% of childhood ALL cases and 75% of infant leukemias. Though current intensive chemotherapy regimens have improved survival in childhood ALL overall, they fail to cure many of these high-risk patient subgroups and inflict significant long-term side effects, demonstrating the urgent need for novel therapies. While exploring the effect of different amino acid depletion strategies, we observed rapid induction of apoptosis upon methionine restriction (MR) in MLLr cells. The histone methyltransferase function of the MLL fusion protein complex requires increased amounts of the methionine metabolite s-adenosylmethionine (SAM) to maintain its hypermethylated state, creating a selective sensitivity of MLLr ALL for perturbations in methionine availability. To explore how MR impinges on MLLr cells' metabolic state we performed global metabolomics on SEM cells and observed rapid effects on metabolism, with 416 metabolites out of the 650 measured differing significantly upon 12 hours of MR. We performed in parallel mRNA sequencing on SEM cells to understand the effect of MR on gene expression and identified 1351 differentially expressed genes. Integrated transcriptomics and metabolomics analysis revealed several key pathways interlinked with SAM, including the folate cycle, polyamine synthesis, arginine/redox metabolism, nucleotide synthesis, and the creatine/urea cycle, with several SAM-dependent enzymes and key byproducts in these pathways being significantly reduced upon MR. Moreover, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells, confirming the critical role of SAM in this leukemia subset. To better understand the effect of methionine and SAM restriction on epigenetic modifications, we looked at histone methylation in a panel of MLLr and non-MLLr B-ALL cell lines and observed a global suppression of methylation upon MR specifically in the pediatric MLLr cells. Additionally, gene expression analysis revealed downregulation of key MLL fusion target genes associated with proliferation and maintenance of the oncogenic phenotype in response to MR, including ZMYND8, ERG, BCL2, PROM1, HMX3, HOXA10, HMGA2, and PBX3. Several of these gene targets are known to be transcriptionally upregulated in MLLr leukemia via aberrant H3K79 methylation. Consistent with this notion, knockdown of the H3K79 demethylase, KDM2B, displayed a more MR-resistant phenotype than its wildtype counterparts. Taken together, MR has a direct effect on global histone methylation in MLLr ALL cells, which in turn disturbs the MLL gene signature critical for their survival. Lastly, immunocompromised mice were engrafted with MLLr SEM cells and treated with a 95% MR diet while leukemia progression was monitored by flow cytometry. The MR diet was well tolerated with minimal weight loss, methionine levels in plasma were roughly 50% reduced compared to controls, and that alone was enough to significantly delay leukemic growth (p = 0.015). While several compounds targeting MAT2A, the enzyme that produces SAM from methionine, are currently in Phase 1 clinical trials, the availability of an FDA approved methionine-free formula for infants allows for rapid translation to the clinic. As a follow-up, we also performed a drug screen using 214 compounds to identify drugs that interact with MR, showing that vinca-alkaloids, including vincristine, strongly synergize. Validation experiments demonstrate that MR triggers a stress induced G2 arrest, priming cells for mitotic spindle destruction with vincristine. In vivo experiments are currently underway using patient-derived xenografts exploring synergistic potential of vincristine with targeting methionine metabolism. In summary, we found that MLLr leukemic cells are selectively vulnerable to perturbations of the methionine cycle due to their increased need for SAM to maintain hypermethylation and aberrant gene activation. Limiting methionine and/or SAM availability provides an attractive adjuvant therapy potentiating the effects of current or novel (targeted) therapies for MLLr leukemia.

The RNA Binding Protein Musashi-2 (MSI2) Sustains the Growth and the Leukemogenic Potential of MLL-Rearranged Acute Lymphoblastic Leukemia, and It Is Involved in Glucocorticoid Resistance

Acute Lymphoblastic Leukemia occurring in infants and carrying the rearrangement of KMT2A, aka MLL (MLLr infant ALL) is a rare but very aggressive leukemia typically associated with poor outcome. Patients suffer from a dismal prognosis (overall survival: 20-40%) mainly due to the resistance to conventional therapy and a high incidence of relapse. The biological mechanisms involved in the pathogenesis and disease progression of infant MLLr leukemia are not completely understood and the identification of druggable genes is urgently needed for the development of novel therapeutic strategies. The RNA-binding protein Musashi-2 (MSI2) is a post-transcriptional regulator of mRNA translation to proteins. MSI2 has a pivotal role in normal hematopoiesis and in a variety of tumors, including leukemia, where it is involved in cell proliferation, differentiation and maintenance of the (cancer) stem cell pool. Although several studies strikingly demonstrated the involvement of MSI2 in chronic and acute myeloid leukemia, with regards to B-ALL, few papers reported over-expression of MSI2 associated with a poor outcome in both pediatric and adult B-ALL patients; however functional data are still missing. By retrospectively analyzing the gene expression profile of a large cohort of Italian patients with pediatric leukemia (n=314) we observed that MSI2 is highly expressed across the different cytogenetic subgroups, and the level of expression is even higher in ALL compared to AML cases; thus reinforcing the hypothesis that MSI2 might have a crucial role also in ALL. To unravel the functional role of MSI2 in MLL-r ALL, we successfully generated a model of MSI2 knock-out (MSI2 KO) using CRISPR/CAS9 genome editing in the MLL::AF4+ ALL cell line SEM. In a long-term competitive assay in vitro, in which the MSI2 KO and CNTRL cells were co-cultured at the initial 90:10 ratio (KO:CNTRL), we observed that after 20 passages in culture the CNTRL counterpart overgrew the MSI2 KO cells (which indeed extinguished); thus demonstrating that the lack of MSI2 leads to a proliferation disadvantage. In agreement with this, MSI2 KO cells show an impaired leukemia-initiating capacity and a lower disease burden in vivo, and mice transplanted with MSI2 KO cells display a prolonged survival compared to controls. Also, we performed a high-throughput drug screening using a manually curated library of 174 compounds (FDA-approved, currently used in the clinic) and we observed that MSI2 KO cells show a marked sensitivity to Glucocorticoids (GCs). Further validation studies in vitro demonstrated that MSI2 KO cells were significantly (100-fold) more sensitive to Prednisolone or Dexamethasone than their MSI2-expressing counterpart. In addition, the treatment of SEM cells (typically resistant to GCs) with the MSI2 inhibitor Ro 08-2750 phenocopied the sensitization to GCs observed in CRISPR-edited MSI2 KO cells, showing synergistic effects in combination with Prednisolone or Dexamethasone. As infant MLLr ALL patients are typically resistant to GCs, our findings suggest that targeting MSI2 might represent a potential novel approach to circumvent the therapy resistance. Finally, by further investigating the biological mechanisms of GC-sensitization, we unraveled a potential role for MSI2 in the regulation of the energetic metabolism of leukemic cells. Indeed, the confocal analysis of mitochondrial membrane potential and the profiling of cellular bioenergetics by Seahorse technology revealed that MSI2 KO cells display mitochondrial hyperactivity at steady-state compared to controls. Upon GCs exposure (which inhibits glycolysis in both MSI2 KO and CNTRL cells) mitochondrial respiration is increased in CNTRL cells to compensate ATP production, while it is severely impaired in MSI2 KO, leading to a mitochondrial crisis and energy failure. The importance of metabolic plasticity of tumor cells in adaptation to stress conditions is emerging as a crucial aspect of cancer biology. The putative role of MSI2 in regulating the energy metabolism of leukemia is a novel finding not previously investigated, which may pave the way for targeting the metabolic vulnerabilities of ALL as a novel therapeutic approach. In conclusion, our study demonstrates that MSI2 sustains the growth in vitro and the leukemogenic potential in vivo of MLLr ALL cells, and it is involved in GC-resistance by regulating the energy metabolism of leukemia.

Interrogating Novel Bromodomain Inhibition Resistance Mechanism in Mllr Leukemia

▪MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood.We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 + leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression.In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia. DisclosuresNo relevant conflicts of interest to declare.

MEIS1 in Hematopoiesis and Cancer. How MEIS1-PBX Interaction Can Be Used in Therapy

Recently MEIS1 emerged as a major determinant of the MLL-r leukemic phenotype. The latest and most efficient drugs effectively decrease the levels of MEIS1 in cancer cells. Together with an overview of the latest drugs developed to target MEIS1 in MLL-r leukemia, we review, in detail, the role of MEIS1 in embryonic and adult hematopoiesis and suggest how a more profound knowledge of MEIS1 biochemistry can be used to design potent and effective drugs against MLL-r leukemia. In addition, we present data showing that the interaction between MEIS1 and PBX1 can be blocked efficiently and might represent a new avenue in anti-MLL-r and anti-leukemic therapy.

Synthesis and Biological Activity of a Cytostatic Inhibitor of MLLr Leukemia Targeting the DOT1L Protein.

Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.

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.

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

Durable Remission after Salvage Chemotherapy with Combining Bortezomib and Vorinostat Followed By CD19 CAR-T Therapy in a Patient with MLL-Rearranged ALL after Early Relapse Post Allogeneic Hematopoietic Stem Cell Transplantation

3D Genomics of Acute Meyloid Leukemia Reveals the Imbalance between DNA Methylation Canyon Interactions and Leukemic Specific Enhancer Network Interactions

Changes in 3D chromatin organization like enhancer hijacking are believed to the driver for disease development like leukemia. Here we performed high-resolution HiC assays on primary acute myeloid leukemia (AML) samples and cell lines to dissect the abnormal 3D chromatin organization in AML. Our data set covers 5 AML samples and 3 AML cell lines. This dataset includes the common genetic abnormalities in AML: MLL-rearrangement, NPM1 mutation, RUNX1 mutation, and IDH1/TET2 mutations. We have recently generated high-resolution map for normal human hematopoietic stem cells (HSC) (Zhang et al. Mole Cell. 2020). In comparison with the HSC 3D chromatin organization, we found TADs and loops are very stable in both primary leukemia samples and cell lines. Less than 5% of all TADs in HSC fuse in AML, mimicking the enhancer hijacking scenario. These fusion events do not cause the gene expression changes of genes in the fused TAD. Interestingly, in TET2 or IDH1 mutated AML blast, two-fold more TAD fusion events occurred in primary AML blast in comparison with RUNX1 and MLL-r leukemia, with a loss in the CTCF sites on the TAD fusion break point. We previously found in HSC, the Polycomb marked DNA methylation Canyons (DMC) form multi-Mb size long-range interactions. DMC interactions in general decrease in primary AMLs. AMLs with IDH1 or TET2 mutations shows the biggest reduction in DMC interactions. Hypermethylation in the DMCs is observed in the AML samples with IDH1/2 or TET2 mutations, suggesting DNA methylation level in DMCs controls DMC 3D interactions directly. In leukemia cell lines, the DMC interactions almost disappear, with further hypermethylation in DMCs. Compared with normal HSC, we found in AML, the AML-specific H3K27ac marked regions form leukemia specific loops and transcription stripes in both cell lines and primary samples. Particularly in MLL-r primary leukemias, we found broad H3K27ac covered, hyperacetylated domains (10kb to 200kb). 22 such hyperacetylated domains were identified and associated with leukemogenic genes such as SATB1, ZEB2 and HOXA. All these domains formed distinct 3D micro TAD in the MLL-r primary leukemia in comparison with the HSPC, and CTCFs are not located at the border of these domains. Taken together, suggest active leukemia specific transcription created new 3D genomic interactions which is independent of cohesion-CTCF mediated loop extrusion. Interestingly, in HOXA cluster, we found a geneless DMC 1.3MB upstream of HOXA switched from Polycomb binding site to active enhancer site in the leukemia cells. By applying CRISPR/Cas9 editing, we found this canyon is essential for survival of HOXA high expressing leukemia cell lines like OCI-AML3 and MV4:11. In summary, we found the 3D chromatin organization in human leukemia significantly alters in two opposite way 1. The significant loss of Polycomb marked DMC interactions caused by the DNA hypermethylation and 2. The leukemic specific hyperacetylated domains form its own distinct micro TAD and stripes in the 3D chromatin organization. Disclosures No relevant conflicts of interest to declare.

Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen.

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

MAT2A as Key Regulator and Therapeutic Target in MLLr Leukemogenesis.

Epigenetic dysregulation plays a pivotal role in mixed-lineage leukemia (MLL) pathogenesis, therefore serving as a suitable therapeutic target. S-adenosylmethionine (SAM) is the universal methyl donor in human cells and is synthesized by methionine adenosyltransferase 2A (MAT2A), which is deregulated in different cancer types. Here, we used our human CRISPR/Cas9-MLL-rearranged (CRISPR/Cas9-MLLr) leukemia model, faithfully mimicking MLLr patients’ pathology with indefinite growth potential in vitro, to evaluate the unknown role of MAT2A. Comparable to publicly available patient data, we detected MAT2A to be significantly overexpressed in our CRISPR/Cas9-MLLr model compared to healthy controls. By using non-MLLr and MLLr cell lines and our model, we detected an MLLr-specific enhanced response to PF-9366, a new MAT2A inhibitor, and small interfering (si) RNA-mediated knockdown of MAT2A, by alteration of the proliferation, viability, differentiation, apoptosis, cell cycling, and histone methylation. Moreover, the combinational treatment of PF-9366 with chemotherapy or targeted therapies against the SAM-dependent methyltransferases, disruptor of telomeric silencing 1 like (DOT1L) and protein arginine methyltransferase 5 (PRMT5), revealed even more pronounced effects. In summary, we uncovered MAT2A as a key regulator in MLL leukemogenesis and its inhibition led to significant anti-leukemic effects. Therefore, our study paves the avenue for clinical application of PF-9366 to improve the treatment of poor prognosis MLLr leukemia.

Pathways, Processes, and Candidate Drugs Associated with a Hoxa Cluster-Dependency Model of Leukemia

High expression of the HOXA cluster correlates with poor clinical outcome in acute myeloid leukemias, particularly those harboring rearrangements of the mixed-lineage-leukemia gene (MLLr). Whilst decreased HOXA expression acts as a readout for candidate experimental therapies, the necessity of the HOXA cluster for leukemia maintenance has not been fully explored. Primary leukemias were generated in hematopoietic stem/progenitor cells from Cre responsive transgenic mice for conditional deletion of the Hoxa locus. Hoxa deletion resulted in reduced proliferation and colony formation in which surviving leukemic cells retained at least one copy of the Hoxa cluster, indicating dependency. Comparative transcriptome analysis of Hoxa wild type and deleted leukemic cells identified a unique gene signature associated with key pathways including transcriptional mis-regulation in cancer, the Fanconi anemia pathway and cell cycle progression. Further bioinformatics analysis of the gene signature identified a number of candidate FDA-approved drugs for potential repurposing in high HOXA expressing cancers including MLLr leukemias. Together these findings support dependency for an MLLr leukemia on Hoxa expression and identified candidate drugs for further therapeutic evaluation.

Melatonin inhibits MLL-rearranged leukemia via RBFOX3/hTERT and NF-κB/COX-2 signaling pathways

MLL-rearranged leukemia is an aggressive malignancy associated with poor outcome, which is refractory to conventional treatment. Melatonin has been proven to exert anti-tumor activity, but the effect of melatonin on MLL-r leukemia and the underlying mechanism remain poorly understood. In this study, melatonin inhibited cell proliferation and induced apoptosis by activating the caspase-dependent apoptotic pathway in MLL-r leukemia cells. Mechanistic investigations revealed that melatonin suppressed the expression of hTERT by abrogating the binding activity of RBFOX3 to the hTERT promoter. Melatonin also blocked NF-κB nuclear translocation and suppressed NF-κB binding to the COX-2 promoter, thereby suppressing the expression of COX-2. In addition, clinical samples revealed that melatonin exerts anti-leukemic activity in primary MLL-r leukemia blasts ex vivo. In vivo, the mice treated with melatonin experienced a larger reduction in leukemic burden than the control group in a MLL-r leukemia xenograft mouse model. Collectively, these results suggest that melatonin inhibits MLL-rearranged leukemia through suppressing the RBFOX3/hTERT and NF-κB/COX-2 signaling pathways. Our findings provide new insights into the role of melatonin for MLL-r leukemia treatment.

Critical Role of Jumonji Domain of JMJD1C in AML Leukemogenesis

MLL-rearranged leukemias are found in 5-10% of adult leukemias and over 70% of infant leukemias and are associated with intermediate to poor prognosis. We have recently shown that JMJD1C, a Jumonji domain containing lysine demethylase (KDM), is important for leukemia stem cell (LSC) function in MLL-AF9 and HOXA9 leukemias but dispensable for normal hematopoietic stem cell function, therefore a potential therapeutic target for acute myeloid leukemia (AML).To determine which domains within JMJD1C are essential for its function in AML, we performed a CRISPR/Cas9 negative selection screen against its coding sequence using guide RNAs (gRNA) tiled at ~ 30bp intervals. Specifically, gRNAs were cloned into a lenti-viral vector with a TdTomato marker gene, transduced into a clonal Cas9 expressing mouse MLL-AF9 leukemia and percentage of gRNA positive cells was analyzed by flow cytometry in a time course. JMJD1C Jumonji and Zinc finger domain (ZFD) and surrounding regions exhibited the most depletion at day 14 compare to day 2, comparable or higher than that of the positive control. To investigate the effect of mutating Jumonji and ZFD on MLL-AF9 leukemogenesis, we subject gRNA transduced MLL-AF9 Cas9 cells to colony forming cell assay. Mutating Jumonji or ZFD impaired colonegenic potential of MLL-AF9 Cas9 cells as evident by a significant reduction in the number of colonies. The resulting colonies appeared smaller and diffused, indicative of cell differentiation. Consistently, we observed impaired proliferation, increased apoptosis and increased expression of the myeloid differentiation marker Mac1 upon mutating these domains. Moreover, mutating Jumonji and ZFD prolonged survival of recipient mice compare to control in transplantation experiment. Finally, we performed chromatin-immunoprecipitation followed by sequencing (ChIP-seq) on H3K27, K36 and K9 methylation in JMJD1C knockout versus floxed MLL-AF9 leukemia cells to identify histone marks that correlate with JMJD1C activity. We observed increased H3K36 trimethylation (me3) level on JMJD1C regulated genes loci in the knockout cells compare to controls. Similar increase was observed upon mutating Jumonji and ZFD using CRISPR/Cas9 in ChIP assay. Using Gene Set Enrichment Analysis (GSEA), we found enrichment of JMJD1C regulated genes in the knockout cells in H3K36me3 ChIP-seq data but not H3K27me3 or H3K9 methylation data. Together, these data suggest that Jumonji and ZFD of JMJD1C are most critical for its function in AML and that methylated H3K36 can be used as a marker for JMJD1C activity at gene loci.Next, we performed single cell RNA-seq to examine the transcriptome change upon mutating the catalytic Jumonji domain. Our analysis showed a hierarchy within control leukemia cells with one cluster bearing more LSC property than the other. Upon mutating Jumonji domain, leukemia cells are driven into differentiated states that bear either more granulocyte or monocyte gene signatures. Using MAST, a single cell transcriptome GSEA tool, we observed enrichment of KRAS signatures in Jumonji mutated cells. This observation in addition to the increased expression of the IL-3 receptor subunit genes Csf2ra/b led us to examine whether the IL-3 signaling pathway is affected in these cells. Western blotting of IL-3 down signaling molecules ERK1/2 and STAT5 showed elevated phosphorylation in Jumonji mutated cells compare to controls both at basal level and in response to IL-3 stimulation. Consistently with this finding, Jumonji mutated cells showed increased sensitivity to inhibitors against RAS (Tipifarnib), MEK (Selumetinib) and JAK (Tofacitinib and Ruxolitinib). Finally, by mining published shRNA and gRNA screen, we observed that mouse as well as human MLLr leukemia cell lines with activating RAS mutations showed decreased or diminished response to knockdown or mutation of JMJD1C suggesting that the presence of RAS mutations render them resistant to JMJD1C modulation.Overall, we show that enzymatic domain of JMJD1C, the Jumonji domain, mediates its function in AML leukemogenesis, therefore can be targeted for potential therapeutic intervention. In addition, we uncovered a novel interplay between JMJD1C and IL-3 signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

MiR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell-like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2-containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.

Abstract 3187: Pediatric Preclinical Testing Consortium evaluation of the menin inhibitor, VTP-50469, against xenograft models of MLL-rearranged infant acute lymphoblastic leukemia

Abstract Introduction: Rearrangements involving the MLL (mixed lineage leukemia, KMT2A) gene (MLLr) occur broadly in acute leukemia, in ~80% of infant acute lymphoblastic leukemia (ALL) cases, and are associated with poor outcome. Menin is a ubiquitously expressed nuclear protein, and the MLLr-menin interaction is the key impetus for transformation of MLLr-expressing cells. VTP-50469 is a potent, orally available, small molecule inhibitor of the MLL-menin interaction with pM binding affinity for menin. Therefore, it was of interest to test the in vivo efficacy of VTP-50469 against preclinical models of infant MLLr-ALL. Methods: Infant MLLr-ALL patient-derived xenografts (PDXs) grew in an orthotopic manner in NSG mice. Engraftment and response to treatment were assessed by enumeration of the % human leukemic blasts in the murine peripheral blood (%huCD45+). Treatment commenced when the median %huCD45+ exceeded 1%, and mice received VTP-50469 (120 mg/kg by oral gavage twice daily x 28) or vehicle. An event was defined as the %huCD45+ >25% or leukemia-related morbidity. The Kaplan-Meier method was used to compare event-free survival (EFS) between treated (T) and control (C) groups. Stringent objective response measures were assigned to each mouse and reported as group medians (Houghton et al, Pediatr. Blood Cancer, 2007;49:928-40). Leukemia infiltration into the femoral bone marrow was also assessed at Day 28 following treatment initiation. VTP-50469 was provided by Vitae Pharmaceuticals. Results: VTP-50469 was well tolerated, with maximum average weight losses of 1.6-6.4% across treatment groups compared to 0-2.0% in vehicle control treated groups. VTP-50469 induced significant differences in EFS distribution compared to control in 6 of 6 (100%) of the evaluable MLLr-ALL PDXs. VTP-50469 T-C values in MLLr-ALL PDXs ranged from 1.2 to 100 days (T/C 1.3-21.1), and Maintained Complete Responses (MCRs) were observed in 5 of 6 PDXs. Two of 8 mice engrafted with an MLLr-ALL harboring the MLL-AFF1 (t4;11) translocation had not reached event >230 days following treatment initiation. A significant reduction (P<0.001) in bone marrow infiltration at Day 28 was observed in 2 of 4 evaluable MLLr-ALL PDXs. VTP-50469 at 30 mg/kg (4-fold lower than its maximum tolerated dose) also elicited MCRs in 8 of 8 mice engrafted with an MLL-AFF1 PDX. The on-target activity of VTP-50469 was verified by its lack of efficacy against an ALL PDX harboring the BCR-ABL1 translocation. Conclusions: VTP-50469 exerted profound in vivo efficacy against ALL PDXs derived from infants harboring MLL-AFF1, MLL-GAS7, and MLL-ENL translocations, and significantly reduced leukemia infiltration in the bone marrow. VTP-50469 was also effective across a broad dose range, indicating that it may represent a novel treatment for MLLr leukemia. (Supported by NCI Grants CA199222 & CA199000) Citation Format: Richard B. Lock, Kathryn Evans, Tara Pritchard, Stephen W. Erickson, Yuelong Guo, David A. Claremon, Gerard M. McGeehan, Beverly A. Teicher, Malcolm A. Smith. Pediatric Preclinical Testing Consortium evaluation of the menin inhibitor, VTP-50469, against xenograft models of MLL-rearranged infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3187.

Piribedil disrupts the MLL1-WDR5 interaction and sensitizes MLL-rearranged acute myeloid leukemia (AML) to doxorubicin-induced apoptosis

Targeting WT MLL for the treatment of MLL-r leukemia, which is highly aggressive and resistant to chemotherapy, has been shown to be a promising strategy. However, drug treatments targeting WT MLL are lacking. We used an in vitro histone methyltransferase assay to screen a library consists of 592 FDA–approved drugs for MLL1 inhibitors by measuring alterations in HTRF signal and found that Piribedil represented a potent activity. Piribedil specifically inhibited the proliferation of MLL-r cells by inducing cell-cycle arrest, apoptosis and myeloid differentiation with little toxicity to the non-MLL cells. Mechanism study showed Piribedil blocked the MLL1-WDR5 interaction and thus selectively reduced MLL1-dependent H3K4 methylation. Importantly, MLL1 depletion induced gene expression that was similar to that induced by Piribedil and rendered the MLL-r cells resistant to Piribedil-induced toxicity, revealing Piribedil exerted anti-leukemia effects by targeting MLL1. Furthermore, both the Piribedil treatment and MLL1 depletion sensitized the MLL-r cells to doxorubicin-induced apoptosis. Our study support the hypothesis that Piribedil could serve as a new drug for the treatment of MLL-r AML and provide new insight for further optimization of targeting MLL1 HMT activity.

Biological characterization of <italic>MLL-NRIP3</italic> fusion gene induced leukemia

Leukemia, originating from hematopoietic stem or progenitor cells, is a malignant proliferative tumor which seriously endangers human health and survival. It is the most common childhood malignancy with an increasing proportion of malignancy in adults. Acute myeloid leukemia (AML) involving MLL (mixed lineage leukemia, MLL-r ) translocation is recognized as an aggressive form of hematopoietic malignancies that exhibit a blockade of differentiation, disruption of genetic and epigenetic regulation, and poor prognosis. Although MLL-NRIP3 fusion gene has been reported recently in establishing leukemia mouse model and its effect on leukemia progression in MLL-r mouse model has also been demonstrated, its leukemia biological characteristics were still not well characterized. Here we investigate the basic biological phenotypes of two MLL-r mouse models ( MLL-AF9 and MLL-NRIP3 ), including: the survival of recipient mice transplanted with leukemia cells; the migration and adhesion abilities of leukocyte cells; the number and the function of leukemia stem cells (LSCs), cell cycle and cell apoptosis, and investigate whether the surface markers of LSCs reported previously are equally applicable to MLL-NRIP3 leukemia cells. The results showed that the survival time of MLL-NRIP3 recipient mice was longer than that of MLL-AF9 . The migration ability of MLL-NRIP3 leukemia cells was less capable than MLL-AF9 , while adhesion was stronger than MLL-AF9 cells. Cell cycle and cell apoptosis of two MLL-r leukemia cells basically had no difference. C olony - forming unit assay and limiting dilution assay both revealed that MLL-AF9 leukemia had a higher number of functional LSCs than MLL-NRIP3 . In summary, MLL-NRIP3 induced leukemia is a low LSCs-enriched MLL-r .