MLL-rearranged Leukemia Cells Research Articles

Non-Canonical Transcription Activator Activity of RNA-Binding Protein MBNL1 Uniquely Controls FLT3 expression in MLL-Rearranged Leukemias

MLL-rearranged (MLL-r) 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 MLL-r patients ultimately relapsed after an initial remission. So far, the molecular mechanism by which MLL-r leukemia maintains progression and prevents differentiation remains largely unclear. Observations in the clinic support aberrant expression and concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 that occur in leukemia, including the MLL-r subtype. To interrogate the novel MLL-r vulnerable genes regulating FLT3, we developed an algorithm so-called “context-dependent association analysis” (CDAA) to explore potential MLL-r dependent FLT3 regulators using the DepMap dataset. The RNA-binding protein MBNL1 was identified as the top candidate. MBNL1 has been recognized as an RNA-binding protein (RBP) involved in splicing, RNA export, and stability. However, our study suggested its non-canonical transcriptional activator function by uniquely controlling FLT3 expression in MLL-r leukemias. CRISPR/Cas9 disruption of the coding region of MBNL1 or CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines. However, the FLT3 gene splicing defect was not observed upon MBNL1 loss, suggesting MBNL1 could be affecting FLT3 expression through a transcriptional regulation mechanism. Genome-wide ChIP-seq assays performed in MLL-r SEM and MOLM13 cells identified only two reproducible MBNL1-bound peaks at a genome-wide scale, one of which was located in the distal enhancer of FLT3, ~170kb away from the FLT3 promoter. CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines without affecting enhancer-promoter looping. Using in vivo assay, SEM cells targeted with sgRNA against the MBNL1-bound site in the distal enhancer of FLT3 transplanted into NSG mice exhibited significant growth retardation. Furthermore, the impaired cell fitness crisis phenotype could be entirely rescued by overexpression of FLT3 cDNA. To identify the protein domains of MBNL1 required for its oncogenic function in MLL-r leukemia cells, an MBNL1 domain CRISPR screen was performed on the SEM cell line, stably expressing Cas9 and a pooled sgRNA library targeting the coding region of MBNL1. Essential domains observed from the screen included MBNL1's zinc finger domains. A biochemistry electrophoretic mobility shift assay (EMSA) was conducted using purified recombinant MBNL1 protein and a 207 bp DNA oligo synthesized based on the MBNL1 ChIP-seq peak at the FLT3 enhancer. The result clearly demonstrated that MBNL1 could directly bind to the enhancer DNA of FLT3 through its zinc fingers 1 and 2 domains, supporting its role as a novel transcriptional regulator of FLT3. Using state-of-art research tools, including genome editing, ChIP-seq, and EMSA assays, we have systematically interrogated an undocumented transcriptional regulation axis of MBNL1/FLT3 in MLL-r leukemias. This work will significantly advance our understanding of how transcription programs and novel factors reconstruct the regulatory network specifying MLL-r dependency, promoting the future development of alternative therapeutic targets for MLL-r leukemia in patients.

Targeting EYA1 Activity in MLL- Rearranged Leukemia

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

Variable Response of MLL-Rearranged Leukemia Cell Lines to Combinations of Menin, CDK9 and DOT1L Inhibitors

Background: MLL (Mixed Lineage Leukemia) rearrangements occur in approximately 10% of leukemias. Infant leukemia has a higher frequency of MLL rearrangement (MLL-r) than leukemia in other age groups. Despite intensified treatment in infant leukemia, outcomes remain poor. MLL-r leukemia depends on the interaction of MLL fusions with menin, DOT1L, and CDK9. Single-agent treatment with menin, DOT1L, and CDK9 inhibitors has shown promising results in preclinical studies, but their clinical efficacy has been limited by primary and acquired resistance. We hypothesized that combination therapy targeting multiple regulators, including menin, DOT1L, and CDK9, would provide a more effective treatment of MLL-r leukemia. Methods: We performed cell viability experiments on leukemia cell lines (MV4;11, MOLM13, RS4;11, THP1, SEM, KOPN8, NOMO1, HL60, ML2) with single, two and three drug combinations using a menin inhibitor (VTP50469), a DOT1L inhibitor (EPZ5676), and a CDK9 inhibitor (AZD4573). The single drug treatment durations with VTP50469, EPZ5676, and AZD4573 were 10 days, 14 days, and 3 days, respectively. The number of viable cells was determined by trypan blue exclusion staining. After the combination treatment, cells were washed with PBS, placed in fresh media for recovery and counted on day 4 or 6. The effectiveness of the combination treatments was determined using SynergyFinder 3.0 web application. Annexin V/PI and CD11b staining were performed using flow cytometry. To demonstrate MLL-menin binding inhibition, we generated an N-terminal FLAG-tagged MLL expression construct in leukemia cell lines and performed co-immunoprecipitation and western blot analysis. Results:We first determined theIC50 values for each inhibitor separately. Based on the IC50 values of VTP50469 (menin inhibitor), we categorized MLL-r cell lines into three groups: very sensitive (MOLM13 and MV4;11), moderately sensitive (RS4;11 and SEM) and resistant (THP1 and ML2) [Fig. A]. Sensitivity followed a similar pattern (MOLM13 and MV4;11 -sensitive, THP1-resistant) when MLL-r cell lines were treated with EPZ5676 (DOT1L Inhibitor). In contrast, AZD4573 demonstrated consistent efficacy across all MLL-r leukemia cell lines, with THP1 and MOLM13 being particularly sensitive with IC50 values in the low nanomolar (nM) range. After identifying the IC50 values of each inhibitor, we performed viability experiments using two and three drug combinations. The combination of VTP50469 (menin inhibitor) and EPZ5676 (DOT1L inhibitor) was superior in decreasing cell viability in MLL-r leukemia cell lines sensitive to VTP50469 (MV4;11, MOLM13 and RS4;11) but not effective in THP1 cells which are resistant to VTP50469 and EPZ5676 [Fig. B]. Synergy scores analyzed using the ZIP model showed additive effects in MOLM13 and a synergistic response in MV4;11 and RS4;11. After 14-17 days of treatment with EPZ5676 combined with VTP50469, MV4;11, MOLM13 and RS4;11 cells exhibited no recovery from treatment, indicating a profound and persistent antileukemia effect of the combination treatment. Conversely, the combination of VTP5069 (menin inhibitor) and AZD4573 (CDK9 inhibitor) showed a synergistic inhibitory effect on cell viability only in THP1 [Fig. B]. Compared to the single-drug treatments, we observed an increased rate of apoptosis and differentiation in THP1 cells treated with the combination of VTP50469 and AZD4573. The three-drug combination of EPZ5676, VTP50469, and AZD4573 was not superior to the two-drug combinations in any of the MLL-r cell lines examined. To characterize VTP50469 resistance, we analyzed the inhibition of MLL-menin binding using an MLL expression construct in VTP50469 sensitive and resistant cell lines treated with VTP50469 doses ranging between 5-500 nM. Following treatment with VTP50469, the level of expression of the MLL construct correlated with the sensitivity of MLL-r leukemia cell lines to the menin inhibitor. Conclusions: Based on our preclinical in vitro findings, the co-inhibition of menin-DOT1L or menin-CDK9 is a promising approach for the treatment of MLL-r leukemia. The varying responses of different MLL-r leukemia cell lines to drug combination treatment indicate that not all cases of MLL-r leukemia will respond uniformly to treatment combinations. Taken together, our data provide a strategy to determine optimal treatment combinations for personalized treatment of MLL-r leukemia.

Telomere Transcription in MLL-Rearranged Leukemia Cell Lines: Increased Levels of TERRA Associate with Lymphoid Lineage and Are Independent of Telomere Length and Ploidy

Telomere transcription into telomeric repeat-containing RNA (TERRA) is an integral component of all aspects of chromosome end protection consisting of telomerase- or recombination-dependent telomere elongation, telomere capping, and the preservation of the (sub)telomeric heterochromatin structure. The chromatin modifier and transcriptional regulator MLL binds to telomeres and regulates TERRA transcription in telomere length homeostasis and response to telomere dysfunction. MLL fusion proteins (MLL-FPs), the product of MLL rearrangements in leukemia, also bind to telomeric chromatin. However, an effect on telomere transcription in MLL-rearranged (MLL-r) leukemia has not yet been evaluated. Here, we show increased UUAGGG repeat-containing RNA levels in MLL-r acute lymphoblastic leukemia (ALL) when compared to non-MLL-r ALL and myeloid leukemia. MLL rearrangements do not affect telomere length and UUAGGG repeat-containing RNA levels correlate with mean telomere length and reflect increased levels of TERRA. Furthermore, high levels of TERRA in MLL-r ALL occur in the presence of telomerase activity and are independent of ploidy, an underestimated source of variation on the overall transcriptome size in a cell. This MLL rearrangement-dependent and lymphoid lineage-associated increase in levels of TERRA supports a sustained telomere transcription by MLL-FPs that correlates with marked genomic stability previously reported in pediatric MLL-r ALL.

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.

Quinazoline-Based Analog of Adenine As an Antidote Against MLL-rearranged Leukemia Cells: Synthesis, Inhibition Assays and Docking Studies

Background: Post-translational modifications of histones constitute a dynamic process impacting gene expression. A well-studied modification is lysine methylation. Among the lysine histone methyltransferases, DOT1L is implicated in various diseases, making it a very interesting target for drug discovery. DOT1L has two substrates, the SAM cofactor that gives the methyl group and the lysine H3K79 substrate. Results: Using molecular docking, the authors explored new bisubstrate analogs to enlarge the chemical landscape of DOT1L inhibitors. The authors showed that quinazoline can successfully replace the adenine in the design of bisubstrate inhibitors of DOT1L, showing similar activity compared with the adenine derivative but with diminished cytotoxicity. Conclusion: The docking model is validated together with the use of quinazoline in the design of bisubstrate inhibitors.

Potent, p53-independent induction of NOXA sensitizes MLL-rearranged B-cell acute lymphoblastic leukemia cells to venetoclax

The prognosis for B-cell precursor acute lymphoblastic leukemia patients with Mixed-Lineage Leukemia (MLL) gene rearrangements (MLLr BCP-ALL) is still extremely poor. Inhibition of anti-apoptotic protein BCL-2 with venetoclax emerged as a promising strategy for this subtype of BCP-ALL, however, lack of sufficient responses in preclinical models and the possibility of developing resistance exclude using venetoclax as monotherapy. Herein, we aimed to uncover potential mechanisms responsible for limited venetoclax activity in MLLr BCP-ALL and to identify drugs that could be used in combination therapy. Using RNA-seq, we observed that long-term exposure to venetoclax in vivo in a patient-derived xenograft model leads to downregulation of several tumor protein 53 (TP53)-related genes. Interestingly, auranofin, a thioredoxin reductase inhibitor, sensitized MLLr BCP-ALL to venetoclax in various in vitro and in vivo models, independently of the p53 pathway functionality. Synergistic activity of these drugs resulted from auranofin-mediated upregulation of NOXA pro-apoptotic protein and potent induction of apoptotic cell death. More specifically, we observed that auranofin orchestrates upregulation of the NOXA-encoding gene Phorbol-12-Myristate-13-Acetate-Induced Protein 1 (PMAIP1) associated with chromatin remodeling and increased transcriptional accessibility. Altogether, these results present an efficacious drug combination that could be considered for the treatment of MLLr BCP-ALL patients, including those with TP53 mutations.

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of SMARCB1, which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of SMARCB1 is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the ‘WIN’ site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation in vitro. These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.

The CDK4/6-UCHL5-BRD4 axis confers resistance to BET inhibitors in MLL-rearranged leukemia cells by suppressing BRD4 protein degradation

Among acute leukemias, mixed-lineage leukemia-rearranged (MLL-r) leukemia is associated with poor prognosis. Bromodomain and extra-terminal inhibitors (BETi) are promising agents for treatment of hematological malignancies; however, the mechanisms underlying sensitivity to BETi and biomarkers to predict sensitivity are yet to be clarified. Here, we established OTX015-resistant MLL-r cell lines (OTX015-R cells) and used them to explore therapeutic targets in BETi-resistant MLL-r leukemia. OTX015-R cells exhibited resistance to various BETi, and levels of bromodomain-containing protein 4 (BRD4) and BRD4-regulated molecules, such as c-MYC and B-cell/CLL lymphoma-2 (BCL-2), were remarkably increased in OTX015-R cells relative to those in the parental cells; however, BRD4 mRNA transcript levels were not elevated. These results suggest that overexpression of BRD4 protein, through suppression of BRD4 degradation, may contribute to BETi-resistance. Notably, expression of ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) was increased in OTX015-R cells. Further, a UCHL5 inhibitor, b-AP15, and UCHL5 knockdown had antitumor effects by degrading BRD4. In addition, sensitivity to OTX015 was partially recovered in OTX015-R cells pretreated with b-AP15. Furthermore, cyclin-dependent kinase 4/6 (CDK4/6) inhibition decreased UCHL5 expression, suppressed OTX015-R cell proliferation, and induced apoptosis. These results indicate that the CDK4/6-UCHL5-BRD4 axis confers resistance to BETi by suppressing BRD4 degradation. We propose that this pathway is a potential novel therapeutic target in BETi-resistant MLL-r leukemia with BRD4 overexpression.

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.

The menin-MLL1 interaction is a molecular dependency in NUP98-rearranged AML

AbstractTranslocations involving the NUP98 gene produce NUP98-fusion proteins and are associated with a poor prognosis in acute myeloid leukemia (AML). MLL1 is a molecular dependency in NUP98-fusion leukemia, and therefore we investigated the efficacy of therapeutic blockade of the menin-MLL1 interaction in NUP98-fusion leukemia models. Using mouse leukemia cell lines driven by NUP98-HOXA9 and NUP98-JARID1A fusion oncoproteins, we demonstrate that NUP98-fusion-driven leukemia is sensitive to the menin-MLL1 inhibitor VTP50469, with an IC50 similar to what we have previously reported for MLL-rearranged and NPM1c leukemia cells. Menin-MLL1 inhibition upregulates markers of differentiation such as CD11b and downregulates expression of proleukemogenic transcription factors such as Meis1 in NUP98-fusion-transformed leukemia cells. We demonstrate that MLL1 and the NUP98 fusion protein itself are evicted from chromatin at a critical set of genes that are essential for the maintenance of the malignant phenotype. In addition to these in vitro studies, we established patient-derived xenograft (PDX) models of NUP98-fusion-driven AML to test the in vivo efficacy of menin-MLL1 inhibition. Treatment with VTP50469 significantly prolongs survival of mice engrafted with NUP98-NSD1 and NUP98-JARID1A leukemias. Gene expression analysis revealed that menin-MLL1 inhibition simultaneously suppresses a proleukemogenic gene expression program, including downregulation of the HOXa cluster, and upregulates tissue-specific markers of differentiation. These preclinical results suggest that menin-MLL1 inhibition may represent a rational, targeted therapy for patients with NUP98-rearranged leukemias.

Discovery of higenamine as a potent, selective and cellular active natural LSD1 inhibitor for MLL-rearranged leukemia therapy

Natural products are a rich source of lead compounds and have shown promise for epigenetic drug discovery. In this work, we discovered higenamine from our natural product library as a potent, selective and cellular active natural LSD1 inhibitor. Higenamine shows acceptable potency against LSD1 and high selectivity towards LSD1 over MAOA/B. Higenamine significantly increases expression of LSD1 substrates H3K4me1 and H3K4me2 in MLL-rearranged leukemia cells MV4-11 and MOLM-13, but nearly had no effect on LSD1 and H3K4Me3. Meanwhile, higenamine dose-dependently suppresses the levels of HOXA9 and MEIS1 that are overexpressed in leukemia cell lines. Notably, higenamine induces cell differentiation of MV4-11 and MOLM-13 cells accompanying by increased expression of CD11b, CD14 and CD86. Higenamine promotes cell apoptosis, inhibits colony formation, but does not inhibit proliferation of leukemia cells significantly. In addition, the expression levels of p53 are dramatically changed by higenamine in an LSD1-dependent manner in MV4-11 cells. Taken together, higenamine could be employed as a starting point for the development of more selective and potent LSD1 inhibitors. Our work firstly reveals the non-classical epigenetic regulation mechanism of higenamine in cancers, and also demonstrates the efficacy of higenamine for MLL-rearranged leukemia therapy.

Small-molecule inhibitor of AF9/ENL-DOT1L/AF4/AFF4 interactions suppresses malignant gene expression and tumor growth.

Chromosome translocations involving mixed lineage leukemia (MLL) gene cause acute leukemia with a poor prognosis. MLL is frequently fused with transcription cofactors AF4 (~35%), AF9 (25%) or its paralog ENL (10%). The AHD domain of AF9/ENL binds to AF4, its paralog AFF4, or histone-H3 lysine-79 (H3K79) methyltransferase DOT1L. Formation of AF9/ENL/AF4/AFF4-containing super elongation complexes (SEC) and the catalytic activity of DOT1L are essential for MLL-rearranged leukemia. Protein-protein interactions (PPI) between AF9/ENL and DOT1L/AF4/AFF4 are therefore a potential drug target.Methods: Compound screening followed by medicinal chemistry was used to find inhibitors of such PPIs, which were examined for their biological activities against MLL-rearranged leukemia and other cancer cells.Results: Compound-1 was identified to be a novel small-molecule inhibitor of the AF9/ENL-DOT1L/AF4/AFF4 interaction with IC50s of 0.9-3.5 µM. Pharmacological inhibition of the PPIs significantly reduced SEC and DOT1L-mediated H3K79 methylation in the leukemia cells. Gene profiling shows compound-1 significantly suppressed the gene signatures related to onco-MLL, DOT1L, HoxA9 and Myc. It selectively inhibited proliferation of onco-MLL- or Myc-driven cancer cells and induced cell differentiation and apoptosis. Compound-1 exhibited strong antitumor activity in a mouse model of MLL-rearranged leukemia.Conclusions: The AF9/ENL-DOT1L/AF4/AFF4 interactions are validated to be an anticancer target and compound-1 is a useful in vivo probe for biological studies as well as a pharmacological lead for further drug development.

Aberrant TRPM4 expression in MLL-rearranged acute myeloid leukemia and its blockade induces cell cycle arrest via AKT/GLI1/Cyclin D1 pathway

Transient Receptor Potential Melastatin Subfamily Member 4 (TRPM4) has been demonstrated to be aberrantly expressed in several cancers but seldom reported in acute leukemia. Based on database mining and validated experiments, our present data show that TRPM4 is selectively overexpressed in AML patients and cell lines with the MLL gene rearrangement. We analyzed the correlation between TRPM4 expression and clinical parameters in a validated cohort of AML patients. Increased TRPM4 expression was associated with significant leukocytosis (p = .028), M4/M5 subtype (p = .000), FLT3-ITD mutation (p = .034), MLL status (p = .007) and a higher risk stratification (p = .001). Knockdown of TRPM4 mediated by siRNA impaired proliferation and arrested the cell cycle at the G0/G1 phase in MLL-rearranged leukemia cells. We suggested that TRPM4 may be involved in the pathogenesis of MLL-rearranged leukemia through regulating the AKT/GLI1/Cyclin D1 pathway. The transcription factor HOXA9 was found to be responsible for upregulation of TRPM4 expression by binding to its promoter. In conclusion, TRPM4 is overexpressed in MLL-rearranged AML and blockade of TRPM4 may be an alternative therapeutic approach in AML patients with high TRPM4 expression.

Effects of triptolide and BET protein inhibitor JQ1 on the proliferation and apoptosis of MLL-rearranged acute myeloid leukemia cells and their mechanisms

Objective To investigate the effects of triptolide (TPL) and BET protein inhibitor JQ1 on proliferation inhibition and apoptosis induction of MLL-rearranged acute myeloid leukemia (AML) cell line MV4-11, and to explore their synergistic mechanisms. Methods MV4-11 cells in logarithmic growth phase were treated with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1, 4 nmol/L TPL or different concentrations of JQ1 combined with 4 nmol/L TPL for 48 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry (FCM), mitochondrial membrane potential was detected by JC-1 method, and expressions of mitochondrial apoptosis pathway-related proteins were detected by Western blot. Results The 50% inhibitory concentration (IC50) value of MV4-11 cells treated with JQ1 for 48 h was (283.9±10.7) nmol/L. However, 4 nmol/L TPL significantly enhanced the inhibitory effect of JQ1 on proliferation of MV4-11 cells, the IC50 value of MV4-11 cells treated with JQ1 combined with TPL was (148.1±2.6) nmol/L, and the difference was statistically significant (t = 25.31, P = 0.029). The result of FCM assay showed that compared with the JQ1 alone group [(9.6±2.3)%, (12.6±1.4)%, (19.5±3.3)%, and (22.7±2.1)%], 4 nmol/L TPL combined with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1 acted on MV4-11 cells for 48 h, the proportions of apoptotic cells were (16.4±1.9)%, (27.5±2.1)%, (32.9±3.6)%, and (35.5±3.0)%, respectively, the difference was statistically significant (F = 9.25, P < 0.01). After treated with 4 nmol/L TPL and JQ1 for 12 h, the level of cell membrane potential in MV4-11 cells was significantly lower than that of JQ1 single agent group, and the difference was statistically significant (P < 0.05). After treated by 4 nmol/L TPL combined with JQ1 for 24 h, the levels of anti-apoptotic proteins bcl-2 and Mcl-1 decreased, and the level of pro-apoptotic protein bax increased. Conclusion TPL can significantly enhance the proliferation inhibition and apoptosis induction effects of BET protein inhibitor JQ1 on MLL-rearranged AML cells, and the mechanism may be related to enhancing the mitochondrial apoptosis pathway. Key words: Gene rearrangement; Leukemia, myeloid, acute; Triptolide; BET protein inhibitor JQ1

Molecular mechanisms by which the mutant ASXL1/BAP1 complex aggravates myeloid leukemia

Mutations in ASXL1, which occur frequently in myeloid neoplasms, often confer poor prognosis. Despite their clinical importance, the precise molecular mechanisms underlying the contribution of mutant ASXL1 to cancer pathogenesis remain to be elucidated. Thus, we analyzed the roles of the hyperactive complex formed by mutant ASXL1 and the deubiquitinase BAP1 in promoting myeloid leukemogenesis. BAP1 expression resulted in the stabilization and increased monoubiquitination of mutant but not wildtype ASXL1. Monoubiquitination of mutant ASXL1 enhanced the catalytic function of BAP1, resulting in a profound reduction in H2AK119ub by counteracting the PRC1 complex. The mutant ASXL1-BAP1 hyperactive complex impaired the multi-lineage differentiation of hematopoietic progenitor cells and accelerated myeloid leukemogenesis. Mechanistically, the mutant ASXL1/BAP1 complex induced the upregulation of HOXA5, HOXA7, HOXA9, and IRF8 via a reduction in H2AK119ub. Importantly, BAP1 depletion inhibited the leukemogenicity of mutant ASXL1-expressing myeloid leukemia cells and MLL-rearranged leukemia cells by reducing the expression levels of HOXA5, HOXA7, and HOXA9. Our findings highlight the potential of BAP1 as a therapeutic target in a broad range of myeloid neoplasms.

ASH2L-Promoted HOXC8 Gene Expression Plays a Role in Mixed Lineage Leukemia-Rearranged Acute Leukemia.

BackgroundMixed lineage leukemia (MLL) fusion protein alone exhibits poor histone lysine methyltransferase (HKMT) activity in catalyzing histone H3 Lys4 trimethylation (H3K4me3) in MLL-rearranged acute leukemia.MethodsTo explore the HKMT effect of another regulatory protein within the complex of proteins associated with Set 1 (COMPASS), we analyzed the H3K4me3 modification of the HOXC8 promoter under the action of ASH2L regulation. Small interfering RNA of ASH2L, chromatin immunoprecipitation, real-time-PCR (RT-PCR), and Western blotting were used to detect the expression of specific regions of the HOXC8 promoter, RBBP5, WDR5, MLL, and BRTF in two MLL-rearranged acute leukemia cell lines (RS4:11 and THP-1 cells).ResultsThe gene and protein expression levels of HOXC8 were significantly downregulated upon treatment with ASH2L-siRNA (as analyzed by targeting specific regions of the HOXC8 promoter located 0 and 3 kb (–3.0 kb) upstream of the transcriptional start site in RSH:11 cells; and –3.0 and –2.0 kb upstream of the transcriptional start site, and +1.4 kb downstream of the transcriptional start site in THP-1 cells). The expression levels of the BRTF, RBBP5, WDR5, and MLL genes were significantly downregulated from the different transcriptional start sites of the HOXC8 promoter in the RSH:11 cell line (P < 0.05). Furthermore, the BPTF and RBBP5 genes were downregulated from the HOXC8 promoter in the THP-1 cell line (P < 0.05).ConclusionBased on these results, we suggest a new concept of histone modification of the ASH2L protein in MLL-rearranged acute leukemia, which cannot carry out methyltransferase activity independently. The protein–protein interactions of ASH2L with other COMPASS members, such as MLL, WDR5, RBBP5, and chromatin remodeling factor BRTF, appear to be essential for its role in the activation of HOXC8 gene transcription.

Rationale for Targeting BCL6 in MLL-Rearranged B-ALL

Background and significance: MLL-gene rearrangements occur in ~10% of B-cell acute lymphoblastic leukemia (B-ALL) and 70% of infant B-ALL and define a group of patients with dismal outcomes. MLL belongs to the family of histone methyltransferases and plays a critical role in maintaining hematopoietic stem cells (Jude et al. 2007). Analyzing gene expression data from 207 children with high-risk ALL, we found that BCL6 is a predictor of poor clinical outcome in B-ALL, particularly in cases with MLL rearrangements. Thus, while the mechanisms of drug-resistance in MLL-rearranged B-ALL are largely unknown, we studied here a potential role of BCL6 in promoting the aggressive and refractory phenotype in this subtype of B-ALL. Results: Immunohistochemical staining of bone marrow biopsies from 10 of 11 MLL-rearranged B-ALL samples studied revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug-resistance in B-ALL (Duy et al. Nature 2011). Our genetic and ChIP-seq analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and upregulated BCL6 expression. While oncogenic MLL-fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to upregulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL-fusions strongly induced transcriptional activation of the pro-apoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small-molecule (FX1) BCL6-inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL-fusions and identified BCL6 as a novel target for the treatment of MLL-rearranged B-ALL. Discussion: Pharmacological inhibition of BCL6 using a retro-inverso peptide inhibitor (RI-BPI) compromised MLL-rearranged B-ALL leukemia-initiation and subverted vincristine-resistance. A central mechanistic aspect of BCL6-function in MLL-rearranged B-ALL involves transcriptional repression of the pro-apoptotic BH3-only molecule Bim (BCL2L11). Profiling for BH3-only proteins in various B-ALL subtypes revealed that MLL-rearranged B-ALL is associated with increased expression of the pro-apoptotic protein BIM (Benito et al. 2015). ABT-199 (venetoclax) is a BH3-mimetic that disrupts the interaction between BCL2 and BIM, leading to BIM release and induction of apoptosis. Previous studies have found that MLL-rearranged leukemia cells are sensitive to the BCL2 inhibitor ABT-199. Besides BCL2, we here identified BCL6 as a central antagonist of pro-apoptotic BIM function in MLL-rearranged B-ALL cells. In genetic experiments, we showed that oncogenic MLL-fusions strongly activated BCL2L11 transcription reinforcing the notion that constitutively high Bim expression levels represents an important and selective vulnerability in MLL-rearranged B-ALL. Both BCL2 and BCL6 represent crucial antagonists of BIM in MLL-rearranged B-ALL, BCL2 mediating BIM-sequestration and BCL6 being required for transcriptional repression of BIM. In support of this scenario, peptide (RI-BPI) and small molecule (FX1) inhibitors of BCL6 strongly synergized with blockade of BCL2-mediated BIM sequestration (ABT-199) in killing MLL-rearranged B-ALL cells. Figure 1 Disclosures Armstrong: AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; Janssen: Research Funding; Novartis: Research Funding; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership. Melnick:Janssen: Research Funding; Constellation: Consultancy; Epizyme: Consultancy. Milne:OxStem Ltd.: Equity Ownership.

Selective Dependency of MLL-Rearranged Leukemia on Immunoproteasome Function

Several cellular pathways control the fine balance between self-renewal and differentiation to maintain leukemia-initiating cell (LIC) function. To identify cellular dependencies with relevance for oncogenic fusion proteins, we performed global proteome profiling. Acute myeloid leukemia (AML) was induced by retroviral expression of either MLL-AF9 (MA9) or AML1-ETO9a (AE) in murine hematopoietic stem and progenitor cells (HSPCs) (Lineage-Sca1+Kit+, LSK) which were subsequently transplanted into irradiated syngeneic recipients. After onset of leukemia, LIC-enriched (GFP+ Kithigh) cells isolated from 4 different primary recipients (per oncogene) were analyzed by in-depth quantitative proteomic analysis using high-resolution mass spectrometry (MS). More than 3,000 proteins were quantified with 868 proteins being differentially expressed between MA9 and AE LIC-enriched populations. In MLL-rearranged (MLLr) cells, gene set enrichment analysis (GSEA) revealed significant enrichment of cellular functions related to protein degradation and proteasome function. As this enrichment is present in MLLr-leukemia but not AE-driven LICs, may indicate an oncogene specific vulnerability. Expression of proteasome subunits is highly heterogeneous between different cell types and therefore may also be influenced by the underlying differentiation stage or oncogenic fusion. In published AML gene-expression datasets, immunoproteasome (IP) subunits PSMB8/LMP7 (p=0.0003***), PSMB9/LMP2 (p=0.0007***) and PSMB10/MECL1 (p&amp;lt;0.0001****) showed significantly higher expression in MLLr compared to non-MLLr-AML. IP is a proteasomal variant constitutively expressed in cells of hematopoietic origin, induced under stimulation with pro-inflammatory cytokines and relevant for mediating stress-responses during inflammation and infection. To assess for functional dependency of MLLr cells on IP subunits we performed an in vitro CRISPR/Cas9 dropout screen in MLLr MOLM-13 cells. Genetic inactivation of PSMB8/LMP7 resulted in outcompetition with 3/5 sgRNAs, while there was less dependency detectable for the other subunits. Specificity of this finding was confirmed in 5 different cell lines (4 MLLr; 1 non-MLLr) by RNAi using 2 shRNAs against PSMB8/LMP7 versus non-targeting control. To confirm these findings in primary cells, we used a previously published conventional LMP7 knockout mouse model (Fehling et al., Science, 1994). LSK cells sorted from the bone marrow (BM) of LMP7 knockout and wildtype mice were retrovirally transformed with either MA9, MLL-ENL (ME) or NUP98-HOXA9 (as non-MLLr control) to assess for disease development by serial plating in methylcellulose. Only in MA9 or ME transformed cells LMP7-deficiency limited re-plating capacity to 2-4 rounds. When we injected 2,5x 104 MA9-infected LSK cells into sublethally irradiated recipient mice, recipients of MA9-LMP7-/- cells (n=12) and MA9-LMP7+/+ (n=12) showed development of AML. However, recipients of MA9-LMP7-/- cells had a significant delay in AML development (median survival 63.0 days for LMP7+/+ versus 92.5 days for LMP7 -/- animals, p=0.0387*). Besides the significant delay in AML development, disease penetrance was reduced by 50%, indicating that deficiency for LMP7 impairs development of MA9 driven AML. In contrast, immunophenotypic abundance of HSPCs in LMP7-/- versus LMP7+/+ animals revealed comparable numbers in all relevant subpopulations. Competitive transplantation of LMP7-/- BM into recipient hosts showed no competitive disadvantage or lack of self-renewal capacity compared to LMP7+/+ controls. Pharmacologic inhibition of IP function using the specific LMP7-inhibitor PR-957 (ONX-0914) resulted in significant delay of disease development in secondary recipient hosts. To assess its effect on LIC frequency we performed limiting dilution assays of MA9 leukemic cells in sublethally irradiated recipient mice. PR-957 treatment reduced LIC frequency compared to DMSO control (1/57410 vs. 1/4450). Pharmacologic inhibition of PSMB8/LMP7 in human MLLr leukemia cell lines induced cellular differentiation. Likewise, cell cycle and metabolism appeared affected, functions which could be confirmed by global transcriptome analysis. Taken together, our studies uncover a selective dependency of MLLr-leukemia on IP function and identify PSMB8/LMP7 as a tractable target. Disclosures Heidel: Celgene: Consultancy; Novartis: Consultancy, Research Funding; CTI: Consultancy.