MLL-rearranged Acute Myeloid Leukemia Research Articles

The epigenetic regulators EP300/CREBBP represent promising therapeutic targets in MLL-rearranged acute myeloid leukemia

Acute myeloid leukemia (AML) with mixed-lineage leukemia (MLL) gene rearrangements (MLL-r) is an aggressive subtype of blood cancer with dismal prognosis, underscoring the urgent need for novel therapeutic strategies. E1A-binding protein (EP300) and CREB-binding protein (CREBBP) function as essential transcriptional coactivators and acetyltransferases, governing leukemogenesis through diverse mechanisms. Targeting EP300/CREBBP holds great promise for treating leukemia with some certain cytogenetic abnormalities. Here, we demonstrated that EP300 and CREBBP are core epigenetic regulators in the pathogenesis of MLL-r AML through assaying the transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Knocking-out EP300/CREBBP and inhibitor (A-485) treatment depressed the MLL-r cells proliferation, while the MLL wild-type cells remained uninfluenced. We found that the CDK4/RB/E2F axis was downregulated specifically in MLL-r AML cell after A-485 treatment by RNA-seq, western blot and cut-tag analyses. EP300/CREBBP inhibitor selectively exerted potent anti-leukemia activity through blocking the MLL-r-BET complex binding to H3K27Ac modification on critical genes loci, distinct from global histone acetylation. Collectively, our study identified EP300/CREBBP as a critical epigenetic driver of MLL-r leukemia and validated their therapeutic potential through targeting inhibition, offering a promising avenue for improving clinical outcomes in this aggressive leukemia.

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.

A novel brain targeting MnOx-based MI-3 nanoplatform for immunogenic cell death initiated high-efficiency antitumor immunity against orthotopic glioblastoma

As the most invasive and lethal primary intracranial malignant tumor, the implementation of immunotherapy to glioblastoma (GBM) is still severely hindered by the limitations of the blood–brain barrier (BBB) and low tumor immunogenicity. Herein, a nanoplatform composed of rabies virus glycoprotein 29 (RVG29)-modified menin-mixed lineage leukemia (MLL) inhibitor (MI-3)-loaded MnOx nanoparticles (MMR NPs) is synthesized for effective GBM therapy through eliciting robust antitumor immune responses. After intravenous injection, MMR NPs can firstly cross the BBB and reach the tumor site due to the existence of RVG29. Then the immunogenic cell death (ICD) of GBM is achieved through MnOx induced reactive oxygen species (ROS) generation and ferroptosis. Meanwhile, MI-3, a specific small molecule inhibitor for MLL-rearranged acute myeloid leukemia, is innovatively applied to GBM treatment and found to further cause robust apoptosis and DNA damage. Collectively, the brain targeted MMR NPs realize high-efficiency tumor growth inhibition and ICD-initiated antitumor immunity activation. This work expands the indications of nanoimmunotherapy to intracranial malignancies and explores the feasibility of transforming “cold” tumors into “hot” tumors strategy for GBM.

Bahcc1 is critical for the aberrant epigenetic program in a mouse model of MLL-ENL–mediated leukemia

AbstractIn leukemogenesis, genotoxic stress in hematopoietic stem and progenitor cells (HSPCs) drives individual context-dependent programs of malignant transformation. In light of the various differentiation stages of HSPCs based on a recently revised definition using CD150/CD48, our analyses showed that a subpopulation of long-term repopulating HSCs was most susceptible to MLL-ENL–mediated transformation. An analysis of the molecular mechanism identified Bromo-adjacent homology domain and coiled-coil containing 1 (Bahcc1), which encodes a reader molecule of trimethylated histone H3 lysine 27 (H3K27me3), as a candidate gene involved in distinct susceptibility to leukemic transformation. Interestingly, Bahcc1 was previously reported to be highly expressed in acute myeloid leukemia (AML) with an unfavorable prognosis, including some cases of MLL-rearranged AML. We found that MLL-ENL upregulated Bahcc1 through binding to its promoter, and that Bahcc1 was involved in MLL-ENL–mediated immortalization at least partly through repression of H3K27me3-marked Cdkn1c. Analyses using bone marrow transplantation in mice showed that depletion of Bahcc1 suppressed the leukemogenic activity of MLL-ENL. In a public database, high BAHCC1 expression was found to be associated with a poor prognosis in pediatric AML, in which BAHCC1 expression was significantly lower in MLL-AF9-AML than in other MLL-fusion-AML. These findings shed light on the distinct immortalization potential of HSPCs and suggest a novel MLL-fusion-Bahcc1 axis, which may lead to development of molecular targeted therapy against MLL-fusion–mediated leukemia.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

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

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

Repressing HIF-1α-Induced HDAC9 Contributes to the Synergistic Effect of Venetoclax and Menin Inhibitor in KMT2Ar AML

Background: MLL-rearranged acute myeloid leukemia (MLLr-AML) is one aggressive subtype of AML, and is conferred one extremely poor prognosis under the current therapeutic strategy. Up to now, it still lacked one effective treatment to maintain the long-term survival for patients with MLLr-AML, while the developments of targeted therapy brought hopes. At present, the star drug BCL-2 inhibitor Venetoclax combined with Azacitidine (AZA) or low-dose Cytarabine (LDAC) has become the first-line scheme for elderly AML, which is not beneficial for MLLr-AML patients. So we are committed to finding a more suitable partner for VEN because MLLr-AML is very sensitive to VEN in vitro studies. The MLL-menin complex is crucial for maintaining MLLr-AML, and the research and application of MLL-menin inhibitors (MEN1i) is currently one of the hotspots in the AML field. Therefore in this study, we aimed to uncover the synergistic effect and mechanism of MEN1i combined with Venetoclax in treating MLLr-AML. Methods: Cell culture and transfection, Cell proliferation, apoptosis and Cell cycle assay, Quantitative RT-PCR, and Western blotting analysis were used to explore biological phenotypes. RNA-seq profiling was performed for DMSO, VEN, MEN1i, and combination of VEN and MEN1i groups in three MLLr-AML cell lines MV4-11, MOLM13, THP-1. Bioinformatic tools were used for differential analysis. Results: MLLr-AML cells were sensitive to VEN or MEN1i monotherapy, and the combination of two drugs had a strong synergistic effect in vitro and in vivo. Furthermore, we found that combination of VEN and MEN1i significantly enhanced apoptotic induction to influence the MLLr-AML survival. To uncover the synergetic mechanism of VEN and MEN1i, we displayed RNA-Sequencing for DMSO, VEN, MEN1i, and combination of VEN and MEN1i groups in three MLLr-AML cell lines MV4-11, MOLM13, THP-1. Finally we found that repressing HIF-1α-induced HDAC9 contributes to the synergistic effect of Venetoclax and MEN1i in MLLr-AML. Conclusion: VEN combined with MEN1i had a strong synergistic effect in the treatment of MLLr-AML. Repressing HIF-1α-induced HDAC9 contributes to the synergistic effect of Venetoclax and MEN1i in MLLr-AML.

Abstract A19: Targeting epigenetic regulators to override cellular identity programs and induce therapeutic differentiation in MLL-rearranged acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a poor-prognosis disease that is universally characterized by a prominent differentiation block. Chemotherapeutic regimens and disease outcomes have not changed in decades, and there is great need for novel therapeutic treatments and approaches. Differentiation therapy is a fundamentally different therapy that aims to reactivate latent maturation programs to induce proliferation arrest and apoptosis. This approach is curative in the promyelocytic AML subtype but not others. It is therefore critical to identify the enforcers of differentiation arrest in non-APL AML. Here, we performed a cell-fate selection CRISPR screen for chromatin factors whose inhibition promotes AML cell differentiation. We found that both genetic and chemical inhibition of the histone acetyltransferase KAT6A induces myeloid maturation, most prominently in MLL-rearranged AML. KAT6A loss markedly reduces self-renewal and extinguishes the proliferative capacity of AML cells. Through ChIP-seq, RNA-seq, and clinical dataset analyses, we found that KAT6A is the likely initiator of a newly-described "writer-reader" module that drives oncogene expression in AML. KAT6A catalyzes promoter H3K9 acetylation, which is bound by the H3K9ac reader ENL, leading to Super-elongation complex recruitment and release of paused RNA PolII at oncogenic loci such as MYC. KAT6A is elevated in human AMLs compared to matched normal tissue, and its downregulation correlates with monocytic differentiation in clinical AML datasets. These findings suggest the potential of targeting KAT6A and/or ENL to disrupt this writer-reader module and ablate downstream MYC transcriptional programs in MLL-rearranged AML. This strategy may be viable in the near future, as a recently developed KAT6A/B inhibitor has already entered clinical trials for breast cancer. In follow-up work, we performed differentiation-specific "synergy screens," in which we delivered sgRNA libraries to U937 cells and ER-Hoxb8 cells treated with or without an LSD1 inhibitor (LSD1i). We identified multiple genes whose knockout synergized with LSD1i treatment to induce complete, terminal differentiation with low toxicity. Genetic results were confirmed with small molecule inhibitors of selected hits, and the most synergistic drug combination was pursued further. We found that AML cells dependent on hyperactivity of both Hoxa9 and Meis1 are most sensitive to this drug combination. Preliminary data suggest that LSD1i treatment alone fails to downregulate Meis1 expression, and that the ability of the second drug to fully ablate Meis1 is the likely mechanism underlying the observed synergy. Importantly, the second drug is also in clinical trials and will be discussed in the abstract presentation. Altogether, this work advances our understanding of the AML differentiation block and nominates a specific drug combination for AML differentiation therapy. Citation Format: Andres Blanco. Targeting epigenetic regulators to override cellular identity programs and induce therapeutic differentiation in MLL-rearranged acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A19.

Abstract 447: Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition

Abstract The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia. Citation Format: Corey Fyfe, Gabriella Fiorentino, Michael J. Steinbaugh, Joana Campos, Hannah Lawson, Jozef Durko, Donal O'Carroll, Kamil Kranc, Darren Harvey, Shomir Ghosh, Robert M. Campbell. Genetic and pharmacological inhibition of TUT4 and TUT7 sensitizes AML cells to BCL-2 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 447.

FBXO22 promotes leukemogenesis by targeting BACH1 in MLL-rearranged acute myeloid leukemia

BackgroundSelectively targeting leukemia stem cells (LSCs) is a promising approach in treating acute myeloid leukemia (AML), for which identification of such therapeutic targets is critical. Increasing lines of evidence indicate that FBXO22 plays a critical role in solid tumor development and therapy response. However, its potential roles in leukemogenesis remain largely unknown.MethodsWe established a mixed lineage leukemia (MLL)-AF9-induced AML model with hematopoietic cell-specific FBXO22 knockout mice to elucidate the role of FBXO22 in AML progression and LSCs regulation, including self-renewal, cell cycle, apoptosis and survival analysis. Immunoprecipitation combined with liquid chromatography-tandem mass spectrometry analysis, Western blotting and rescue experiments were performed to study the mechanisms underlying the oncogenic role of FBXO22.ResultsFBXO22 was highly expressed in AML, especially in MLL-rearranged (MLLr) AML. Upon FBXO22 knockdown, human MLLr leukemia cells presented markedly increased apoptosis. Although conditional deletion of Fbxo22 in hematopoietic cells did not significantly affect the function of hematopoietic stem cells, MLL-AF9-induced leukemogenesis was dramatically abrogated upon Fbxo22 deletion, together with remarkably reduced LSCs after serial transplantations. Mechanistically, FBXO22 promoted degradation of BACH1 in MLLr AML cells, and overexpression of BACH1 suppressed MLLr AML progression. In line with this, heterozygous deletion of BACH1 significantly reversed delayed leukemogenesis in Fbxo22-deficient mice.ConclusionsFBXO22 promotes MLLr AML progression by targeting BACH1 and targeting FBXO22 might be an ideal strategy to eradicate LSCs without influencing normal hematopoiesis.

The Mechanism of Artesunate Combined with Cytarabine and/or Daunorubicin on the Apoptosis of MV4-11 MLL-rearranged Acute Myeloid Leukemia Cell Line

To investigate the effect and mechanism of artesunate (ARTS) combined with cytarabine(Ara-C) and/or daunorubicin (DNR) on the proliferation and apoptosis of MV4-11 human mixed-lineage leukemia rearranged（MLL-r） acute myeloid leukemia (AML) cell line. CCK-8 assay was used to detect the proliferation effect of individual or in combination of ARTS, DNR, Ara-C on MV4-11 cells. The IC50 of ARTS, DNR and Ara-C was calculated separately. The cell apoptosis and expression of receptors DR4 and DR5 were detected by flow cytometry. Western blot was used to detect the expression of Caspase-3 and Caspase-9 in each groups. The inhibition effect of ARTS, Ara-C and DNR on the proliferation of MV4-11 were all dose-dependently (r=0.99, 0.90 and 0.97, respectively). The IC50 of ARTS, Ara-C and DNR on MV4-11 for 48 hours were 0.31 μg/ml, 1.43 μmol/L and 22.47 nmol/L, respectively. At the dose of ARTS 0.3 μg/ml， Ara-C 1.0 μmol/L and DNR 15 nmol/L, the proliferation rate for 48 hours of the tri-combination treatment was significantly lower than that of the bi-combination treatment, while both were significantly lower than that of the individual treatment (all P＜0.05). In terms of bi-combination treatment, the cells proliferation rate for 48 hours of the ARTS+Ara-C group was significantly lower than that of the ARTS+DNR group, while both were significantly lower than that of the Ara-C+DNR group (all P＜0.05). The cooperativity index (CI) of bi- and tri-combination treatment were all less than 1. After 48 hours of drug action, the cell apoptosis rate of the ARTS+DNR+Ara-C group was significantly higher than that of the Ara-C+DNR group, while both were significantly higher than that of the ARTS+DNR group (all P＜0.05). Meanwhile, the was no statistical difference between the cells apoptotic rate of the ARTS+DNR+Ara-C group and the ARTS+Ara-C group (P>0.05). The expression of DR4 and DR5 also showed no difference between control group and drug group. Compared with the DNR+Ara-C group, the expressions of Caspase-3 were significantly down-regulated in both the ARTS+DNR+Ara-C group and the ARTS+Ara-C group (all P＜0.05). The down-regulation of Caspase-3 expression was the most significantly in the combination group of three drugs, while the Caspase-9 expressions in different groups showed no apparent change. The in vitro study showed that tri-combination of ARTS+Ara-C+DNR and bi-combination of ARTS+Ara-C could inhibit the proliferation and promote apoptosis of MV4-11 cell line. The inhibition effect of these two combinations were significantly superior to that of the traditional Ara-C+DNR treatment. The mechanism underlying this finding may be identified by the down regulation of Caspase-3, while no altered expression was observed of Caspase-9, DR4 and DR5.

Genetic and Pharmacological Inhibition of TUT4/TUT7 Sensitizes AML Cells to BCL-2 Inhibition

The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia.

Retrospective Lineage Tracing of Pediatric Acute Myeloid Leukemia Using Single-Cell Whole Genome Sequencing

Background Acute myeloid leukemia (AML) accounts for 15-20% of all pediatric acute leukemias and is a very heterogeneous disease, both genetically and phenotypically. Although survival has increased gradually during the last decades, relapse-rates remain around 20-30%, and children who relapse have a relatively poor outcome. Still, the exact cause of pediatric AML (pAML) remains unclear. Previously, we found an increased clonal mutation burden in leukemic blasts of a subset of pAML cases, including the ones characterized by RUNX1-RUNX1T1 translocations. Here, we study the mutation accumulation and clonal evolution of pAML to elucidate the timing of driver accumulation and mutational processes underlying leukemogenesis in children. Methods All pAML bone marrow samples were acquired via the biobank of the Princess Máxima Center for pediatric oncology after ethical approval was obtained. We used primary template-directed amplification (PTA) to accurately analyze the complete genomes of single cells at nucleotide resolution. For PTA-mediated whole genome sequencing (WGS), we sorted single leukemic blasts, hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs) and several differentiated cell types. Subsequently, WGS data was used to study mutation accumulation and to perform phylogenetic lineage tracing of different cell types of the same patient. Results Thus far, we have analyzed the single-cell WGS data of 18 cells from two AML samples characterized by NUP98-NSD1 and RUNX1-RUNX1T1 fusions. We identified multiple cells that immunophenotypically resembled HSCs, MPPs and more mature cell types from different lineages, that shared the fusion gene with the AML blasts (Figure 1). Moreover, a subset of these cells shared one or more additional driver mutations with the immunophenotypically defined bulk leukemic blasts. Subsequent mutation analysis showed that cells with the oncogenic fusion had an increased somatic mutation burden compared to cells without the fusion. Accordingly, we studied mutational signatures to identify the processes underlying this mutation burden. Interestingly, besides the expected mutational signatures, we found that the increased mutation burden was mainly characterized by SBS18, a signature that is associated with damage induced by oxidative stress. This process was active throughout the leukemic lineage, whereas unrelated HSCs and MPPs of the same patients did not display this signature. This finding indicates that the process was specific for the leukemic lineage and therefore likely cell intrinsic. Next, we used driver and passenger mutations to construct phylogenetic lineage trees and found that the accumulation of drivers occurred stepwise. We show that the oncogenic fusion was the first driving event in different patients, followed by accumulation of additional driver mutations. For example, in one patient, based on the clock-like mutation burdens in the separate branches, we could estimate that the first drivers were already present when the patient was between 7.5 to 9 years old (Figure 1, branch A), while this patient was diagnosed at 15.2 years. We have previously identified similar patterns in multiple MLL-rearranged therapy-related AML (t-AML) patients. Both in the t-AML and pAML cases, some leukemic blasts did not have any additional genetic drivers compared to the phenotypical stem and progenitor cells. Conclusions We used single-cell WGS to identify pre-leukemic cells in bone marrow samples of pAML patients, which shared both the oncogenic rearrangement and additional driver mutations with the AML blasts. These pre-leukemic cells showed an increased mutation load, caused by distinct mutational processes. In addition, these cells were immunophenotypically and functionally stem-cell like, contributing to hematopoiesis. Our analyses show that, despite their aberrations, these cells still have differentiation potential. Interestingly, the first driver aberrations of pAML originated years before diagnosis. These data suggest that genetic driving aberrations may not be the only underlying mechanism of AML development in children. Noncoding drivers, epigenetic changes or environmental influences may play an important role in the final steps towards leukemogenesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Small-Molecule Inhibition of the Acyl-Lysine Reader ENL as a Strategy against Acute Myeloid Leukemia.

AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.

Abstract CT210: A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, as single-agent in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM)

Abstract Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 demonstrated a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, DLBCL lines representing Double/Triple Hit Lymphoma (DHL/THL), Double Expressor Lymphoma (DEL), and MM cell lines harboring diverse mutational backgrounds. BMF-219 also exhibited high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and R/R MM. Methods: BF-MNN-101 (NCT05153330) is a prospective, open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of oral BMF-219 administered once daily in patients with R/R AL, DLBCL and MM who have received standard therapy. Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication following a modified Fibonacci sequence until 1 subject experiences either a ≥ Grade 2 adverse event or dose limiting toxicity (DLT). At that point, the current and all subsequent dose level(s) for the specific indication/cohort will follow a classical “3 + 3” design. Each treatment cycle will be 28 days until progression or intolerability occurs. Following conclusion of the parallel dose-escalations, expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data. Eligible patients include those with R/R AL, R/R DLBLC ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies who failed or are ineligible for any standard therapies. Patients must have ECOG ≤2, and adequate organ function with prior treatment-related toxicities resolved to ≤ Grade 2. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease. The primary objective is to determine independently for each cohort/indication the optimal biological dose (OBD)/recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels in the escalation phase and during the expansion cohort portion of the study. The first patient in the study is anticipated to dose in Q1 of 2022. Citation Format: Farhad Ravandi-Kashani, Ashwin Kishtagari, Hetty Carraway, Emily Curran, Gary Schiller, Alex Cacovean, Bhagyashree Yadav, Thomas Butler, Jeffrey Lancet. A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, as single-agent in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT210.

Abstract 681: Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition

Abstract MLL1 (KMT2A) translocations lead to aberrant expression of stem cell associated gene programs in hematopoietic cells producing a particularly aggressive subtype of leukemia, namely MLL-rearranged acute myeloid and lymphoblastic leukemia (AML and ALL, respectively). In pediatric leukemia, the most common rearrangement is the t(9; 11) (p22; q23) reciprocal translocation, which results in the expression of the MLL-AF9 fusion gene. Since the MLL-AF9 rearrangement does not lead to direct activation of an enzyme, small molecule drug discovery remains challenging and despite decades of research on MLL-rearranged leukemia, we have been limited by the inability of acutely assessing the consequences of direct MLL-AF9 inactivation in relevant model systems. By coupling rapid PROTAC-mediated degradation of the oncogenic MLL-AF9 fusion protein with genome-wide gene expression and chromatin analyses we have now been able to identify the immediate transcriptional and chromatin state consequences of MLL-AF9 degradation. Specifically, we have established a core set of MLL-AF9 target genes whose expression changes within minutes of MLL-AF9 degradation. These rapid expression changes are mediated by changes in productive RNA Polymerase II elongation and increased RNA Polymerase II pausing. At later timepoints, degradation of the fusion protein also induces loss of an active chromatin landscape at MLL-AF9 target genes, characterized by loss of MLL-AF9 associated proteins and activating histone modifications. These insights improve our mechanistic understanding of how MLL-AF9 mediates chromatin remodeling and leukemogenesis, while also helping us evaluate small molecule inhibitors of epigenetic mechanisms that target the MLL-AF9 protein complex. Specifically, we investigated how degradation of MLL-AF9 compares to enzymatic inhibition of DOT1L or inhibition of the MENIN-MLL interaction. Interestingly, both DOT1L and MENIN inhibitors fell short when comparing their effects to MLL-AF9 degradation and only combined DOT1L/MENIN inhibition induced global MLL-AF9 destabilization on chromatin, thus disrupting the full MLL-AF9 directed epigenetic and transcriptional program. Ultimately, we were able to confirm these findings in a patient-derived xenograft model in vivo, where only the combination was able to significantly prolong leukemia free survival. In summary, our studies establish the cellular and molecular consequences of targeted degradation of the MLL-AF9 fusion oncoprotein and help improve our understanding of small molecule inhibitors of epigenetic mechanisms as cancer therapies. Citation Format: Sarah Naomi Olsen, Laura Godfrey, James P. Healy, Charles Hatton, Scott A. Armstrong. Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 681.

COVALENT-101: A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM).

TPS7064 Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair, plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is therefore a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable, small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 exhibited a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, DLBCL lines representing Double/Triple Hit Lymphoma (DHL/THL) and Double Expressor Lymphoma (DEL), and MM cell lines with diverse mutational backgrounds. BMF-219 also showed high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and R/R MM. Methods: COVALENT-101 (NCT05153330) is an open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of once daily oral BMF-219 in patients with R/R AL, DLBCL and MM who have received standard therapy. Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication until 1 subject experiences either a ≥ Grade 2 related-adverse event or dose limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 + 3” design. Treatment will continue in 28-day cycles until progression or intolerability. Expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data. Patients with R/R AL, R/R DLBCL ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies and failed or are ineligible for any standard therapies are eligible. Patients must have ECOG PS ≤ 2, and adequate organ function. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease. The primary objective is to determine independently for each cohort/indication the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels. The enrollment commenced in January 2022. Clinical trial information: NCT05153330.

JMJD1C-regulated lipid synthesis contributes to the maintenance of MLL-rearranged acute myeloid leukemia

Mixed Lineage Leukemia rearranged acute myeloid leukemia (MLLr AML) predicts a poor prognosis. Histone demethylase JMJD1C is a potential druggable target of MLLr AML. However, little is known about how JMJD1C contributes to MLLr AML. Here we found that JMJD1C regulates lipid synthesis-associated genes including FADS2, SCD in MLLr AML cells. Lipid synthesis-associated protein FABP5 was identified as a specific interacting protein of JMJD1C and binds to the jumonji domain of JMJD1C. FABP5 also regulates JMJD1C mRNA and protein expression. JDI-10, a small molecular inhibitor of JMJD1C identified by us, represses MLLr AML cells, induces apoptosis, and decreases JMJD1C-regulated lipid synthesis genes. Moreover, JDI-10 mediated suppression of MLLr AML cells can be rescued by palmitic acid, oleic acid, or recombinant FABP5. In summary, we identified that JMJD1C-regulated lipid synthesis contributes to the maintenance of MLLr AML. Lipid synthesis repression may represent a new direction for the treatment of MLLr AML.

Essential Roles of Transcription Factor MEF2D in the Maintenance of MLL-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease with uncontrolled proliferation of myeloid progenitor cells and a failure of proper cell differentiation. Chromosomal translocations of the KMT2A (MLL) gene are found in about 10% AMLs affecting both children and adults. Patients harboring MLL rearrangement (MLL-r) have a high relapse rate and poor overall survival, emphasizing an unmet need to understand MLL-r AML pathogenesis and develop new therapeutic means. Through a comparative analysis of super-enhancers in AML cell lines and primary samples, we discovered that MLL-r leukemia cells preferentially gain super-enhancer signals at the MEF2D gene. MEF2D is aberrantly activated in MLL-r AML patient samples and predicts poor disease outcomes, implying a potential oncogenic role of MEF2D. Indeed, depletion of MEF2D inhibits human MLL-r leukemia growth and induces profound differentiation. Mechanistically, MEF2D directly represses CEBPE, a master regulator of myeloid differentiation. CEBPE depletion could rescue the growth defect and cell differentiation induced by MEF2D loss. We also demonstrated that MEF2D is positively regulated by HOXA9, and downregulation of MEF2D is an important mechanism for DOT1L inhibitor-induced anti-leukemia effects. Interestingly, MEF2C, a target of MLL fusion oncoproteins, is by far the most studied MEF2 family member with essential roles in MLL-r AML. Our genetic, biochemical, and integrative genomic data shows that MEF2D and MEF2C, both upregulated in MLL-r AML, interact with each other, colocalize genome-widely, and co-regulate target gene expression. We are currently investigating the molecular mechanisms underlying the interdependent function between MEF2 paralogs in MLL-r AML. Collectively, our findings suggest that MEF2D is a novel transcriptional dependency in MLL-r AML and uncover the MEF2-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block. DisclosuresNo relevant conflicts of interest to declare.

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.