EVI1 Overexpression Research Articles

Aberrant ecotropic viral integration site-1 (EVI-1) and myocyte enhancer factor 2 C gene (MEF2C) in adult acute myeloid leukemia are associated with adverse t (9:22) & 11q23 rearrangements

Acute myeloid leukemia (AML) shows multiple chromosomal translocations & point mutations which can be used to refine risk-adapted therapy in AML patients. Ecotropic viral integration site-1 (EVI-1) & myocyte enhancer factor 2 C gene (MEF2C) are key regulatory transcription factors in hematopoiesis and leukemogenesis & both drive immune escape.This prospective study involved 80 adult de novo AML patients recruited from Oncology Center, Mansoura University, between March 2019 and July 2021. The MEF2C and EVI1 expression were measured using a Taqman probe-based qPCR assay.The results revealed that EVI1 and MEF2C expression were significantly elevated in AML patients as compared to control subjects (p = 0.001. 0.007 respectively). Aberrant expressions of EVI1 and MEF2C showed a significant negative correlation with hemoglobin levels (p = 0.034, 0.025 respectively), & bone marrow blasts (p = 0.007, 0.002 respectively). 11q23 translocation was significantly associated with EVI1 and MEF2C (p = 0.004 and 0.02 respectively). Also, t (9;22) was significantly associated with EVI1 and MEF2C (p = 0.01 and 0.03 respectively), higher expression of EVI1 and MEF2C were significantly associated with inferior outcome after induction therapy (p = 0.001 and 0.018 respectively) and shorter overall survival (p = 0.001, 0.014 respectively).In conclusion, EVI1 & MEF2C were significantly expressed in AML cases. EVI1 & MEF2C overexpression were significantly associated with 11q23 rearrangements and t (9;22) and were indicators for poor outcome in adult AML patients; These results could be a step towards personalized therapy in those patients.

Abstract 3351: Identifying novel therapies from unbiased screens in AML with MECOM re-arrangement

Abstract EVI1 is a transcriptional regulator encoded from the MECOM locus (3q26.2). Aberrant EVI1 expression is observed in ~10% of de novo AML, of which about half are due to inv3(q21;q26.2) or t(3;3)(q21;q26.2) chromosomal rearrangements [3q26.2-r]. 3q26.2-r AMLs generally have a stem-like phenotype, are refractory to current therapy options and exhibit poor overall survival. Therefore, there is an unmet need to develop novel targeted therapies with improved efficacy in repressing EVI1 and its targets, thereby improving outcomes in AML with EVI1 overexpression. We conducted an unbiased high-throughput drug screen, utilizing a library of mechanistically annotated drugs (NCATS Mechanism Interrogation Plates [MIPE 5.0]), against seven AML cell lines (3q26.2-r: UCSD-AML1, HNT-34, AML191, AML194; non-3q26.2-r AML: SET-2, MV4-11, OCI-AML3). This exploited the mechanistic redundancy in the library to identify druggable, target-level dependencies in 3q26.2-r AML, exhibiting absolute potency and relative activity as compared to other AML subtypes. BRD4 was identified as an absolute dependency in 3q26.2-r AMLs. An unbiased epigenetic modifier CRISPR screen in UCSD-AML further confirmed BRD4 as a dependency, confirming previous reports of BET inhibition (BETi) efficacy in 3q26.2-r AML. Comparison of 3q26.2-r versus other AML cell lines also identified XIAP, mTOR, PIK3CA and Bcl-xL as druggable vulnerabilities in 3q26.2-r AML. In follow-up experiments, IAP family inhibitors/SMAC mimetics induced significantly more dose-dependent apoptosis in 3q26.2-r versus the other AML cell lines, which was associated with reduction of IAP proteins, p-ERK, MCL1 and Bcl-xL, but increase in levels of cleaved caspase-3 and PARP. BETi (mivebresib) also induced dose-dependent apoptosis, and reduced EVI1, c-Myc, c-Myb, IAP proteins, and CDK4/6, while increasing levels of HEXIM1 and cleaved PARP. Consistent with this, co-treatment of mivebresib with SMAC mimetic birinapant or IAP inhibitor LCL161 was synergistically lethal against 3q26.2-r cells. Co-targeting the other identified druggable vulnerabilities from the drug screen with BETi (mTOR/PI3K with BGT-226 and dactolisib, Bcl-xL with navitoclax and A1155463, as well as CBP/p300 (with GNE781, identified through the CRISPR screen), exerted synergistic lethality in 3q26.2-r AML cells. Finally, in a xenograft model of 3q26.2-r AML in immune-depleted mice, while monotherapy with LCL161 or dactolisib significantly reduced the AML burden with minimal toxicity, co-treatment with mivebresib and LCL161 or dactolisib was superior in reducing AML burden in the xenograft model. These findings demonstrate promising preclinical activity of BETi and IAP protein or mTOR/PI3K antagonists in the cellular models of AML with EVI1 overexpression. This supports the rationale to determine in vivo efficacy of these BETi-based combinations against this therapy-resistant AML sub-type. Citation Format: Christine E. Birdwell, Warren C. Fiskus, Christopher P. Mill, Tapan M. Kadia, Naval Daver, Courtney D. DiNardo, Koji Sasaki, John A. Davis, Kaberi Das, Hanxi Hou, Michele Cerebelli, Kapil N. Bhalla. Identifying novel therapies from unbiased screens in AML with MECOM re-arrangement [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 3351.

Abstract 3040: Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer

Abstract Epithelial ovarian cancer (OC) is typically diagnosed at a late stage and, despite treatment with cytoreductive surgery and chemotherapy, 70-80% of patients relapse and eventually die due to the disease. Major challenges to improve patient outcomes are to overcome therapy resistance and to individually tailor therapy. Uncovering the molecular drivers of OC, especially in the most common subgroup high-grade serous (HGS), will help develop novel therapeutic strategies to meet these challenges. Using the Cancer Genome Atlas (TCGA) data, we observed that a locus in 3q26.2, near the EVI1 oncogene, was the most significantly amplified region in HGS OC, occurring in more than 80% of those tumors. We validated these findings in an OC dataset from the Hartwig Medical Foundation and in OC cell lines from the DepMap database. Furthermore, HGS OC had the highest mean copy number of the 3q26.2 locus co-occurring with high mean EVI1 expression among all tumor entities in the pan-cancer TCGA dataset. These EVI1 expression levels are significantly higher than in normal fallopian tube, from which HGS OC most likely arises. EVI1 overexpression promotes leukemogenesis in 3q26-rearranged acute myeloid leukemia (AML), as a result of chromosomal translocations that enable hijacking of hyperactive enhancers. Here, we hypothesize that EVI1 also plays a crucial role in OC development, but that its aberrant expression in this disease is driven by the amplification of yet undiscovered regulatory elements near EVI1. We showed that EVI1-positive OC cell lines, of which the majority are HGS, are EVI1-dependent and harbor a ~200 kb amplified region near EVI1. This region interacts with the EVI1 promoter (4C-Seq), is characterized by open chromatin (ATAC-Seq), and shows high levels of H3K27 acetylation (H3K27ac ChIP-Seq), suggesting the presence of an active enhancer. Furthermore, based upon H3K27ac signal, we identified within this region a super-enhancer of approximately 65 kb. In OC cell lines without detectable EVI1 levels or cell lines from other cancer subtypes, such as breast and colon cancer, this location had no active enhancer and did not loop to the EVI1 promoter. Altogether, these data point to the generation of a hyperactive super-enhancer driving EVI1 overexpression in OC. These findings emphasize the importance of epigenetic control in the regulation of EVI1 expression, as previously shown in 3q26-rearranged AML. Since OC tumor cell lines are strongly dependent on EVI1, interference with this super-enhancer, leading to loss of EVI1 expression, could be exploited to inhibit tumor growth. For this purpose, we generated GFP-tagged EVI1 cell line models (e.g. SKOV3), which will be used to discover small molecules to target the activity of this super-enhancer. In conclusion, we discovered a new mechanism of enhancer-mediated overexpression of the EVI1 oncogene as a molecular driver in ovarian cancer, with possible implications for therapy. Citation Format: Leonie Smeenk, Sabrina Kruse, Roger Mulet-Lazaro, Ingrid Boere, John Martens, Stefan Gröschel, Claudia Scholl, Stefan Fröhling, Ruud Delwel. Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer [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 3040.

The role and regulation of EVI1 in normal hematopoiesis and hematopoietic malignancies

EVI1 is a zinc finger transcription factor encoded by the MECOM locus and is essential for the development and maintenance of hematopoietic stem cells. However, overexpression of EVI1 in various myeloid malignancies is associated with aggressive clinical behavior and poor outcome. The locus encodes multiple isoforms that are differentially acting and independently regulated. EVI1 interacts with a variety of transcription and epigenetic factors via different domains. It also regulates cell survival, differentiation, and proliferation through a variety of mechanisms, including transcriptional activation and repression, regulation of other transcription factors' activity, and chromatin remodeling. While the mechanism by which 3q26 translocation leads to high EVI1 expression through enhancer hijacking of genes active in myeloid development is now better understood, regulation of EVI1 expression in the absence of chromosomal translocations and in normal hematopoiesis remains unclear. Recent studies have provided insight into the regulatory mechanisms of EVI1 expression and action, which may lead to development of targeted therapies in the near future.

Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression.

AML with chromosomal alterations involving 3q26 overexpresses the transcription factor (TF) EVI1, associated with therapy refractoriness and inferior overall survival in AML. Consistent with a CRISPR screen highlighting BRD4 dependency, treatment with BET inhibitor (BETi) repressed EVI1, LEF1, c-Myc, c-Myb, CDK4/6, and MCL1, and induced apoptosis of AML cells with 3q26 lesions. Tegavivint (TV, BC-2059), known to disrupt the binding of nuclear β-catenin and TCF7L2/LEF1 with TBL1, also inhibited co-localization of EVI1 with TBL1 and dose-dependently induced apoptosis in AML cell lines and patient-derived (PD) AML cells with 3q26.2 lesions. TV treatment repressed EVI1, attenuated enhancer activity at ERG, TCF7L2, GATA2 and MECOM loci, abolished interactions between MYC enhancers, repressing AML stemness while upregulating mRNA gene-sets of interferon/inflammatory response, TGF-β signaling and apoptosis-regulation. Co-treatment with TV and BETi or venetoclax induced synergistic in vitro lethality and reduced AML burden, improving survival of NSG mice harboring xenografts of AML with 3q26.2 lesions.

Acute Myeloid Leukemia with Inv(3) or t(3;3): A Clinical and Cytogenetic Characterization of 40 Patients

Background: Acute myeloid leukemia (AML) with inv(3) or t(3;3) is an extremely aggressive and rare subtype of AML. Leukemogenesis in inv(3) AML is in part due to juxtaposition of the GATA2 enhancer in close proximity to the MECOM/EVI1 promoter; as the result, the fusion drives EVI1 overexpression and uncontrolled proliferation of myeloid progenitors. Given its rarity, published clinical outcome data are limited. To improve our understanding of the clinical outcomes of inv(3) AML, we conducted a retrospective study to describe the clinical features of a cohort of newly diagnosed inv(3)/t(3;3) AML patients treated at Stanford Cancer Center between 2002 and 2023. Methods: Data were retrospectively collected from databases associated with the Stanford Health Care electronic health record (EHR), and diagnosis dates came from the Stanford Cancer Registry. Inv(3)/t(3;3) AML diagnosis was defined according to WHO criteria as the presence of ≥20% blasts in the bone marrow (BM) or peripheral blood (PB) and the presence of inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2) on cytogenetic analysis at initial diagnosis. Response to first induction, salvage, and consolidation therapy as well as allogeneic hematopoietic stem cell transplant (alloHSCT) were captured. CR was defined as the presence of <5% of blasts in the BM evaluation by morphological evaluation after each line of treatment. Overall survival (OS) was defined as time from diagnosis to death from any cause. A descriptive study of the main demographic, clinical, hematologic, and cytogenetic variables of patients who received intensive and non-intensive therapy was performed. The analysis was performed using R 4.3.1. A p value of < 0.05 was defined to be statistically significant. Results: A total of 40 patients met inclusion criteria including 68% (n=27) with de novo AML, 25% (n=10) with secondary AML from an antecedent hematological disorder and 7.5% (n=3) therapy-related AML. Clinical characteristics of the patients are summarized in Figure 1A. The median age at time of diagnosis was 56 (range, 19-78). There was a slight female predominance, with 58% (n=23) female and 42% (n=17) males. Ninety-three percent of patients had an ECOG score ≤1 (n=37). Median leukocyte, hemoglobin, and platelet counts were 4.5 K/uL, [1.3-761], 9.4 g/dL, [3.4-13.6], and 98 K/uL [9-592], respectively. The median blast percentage in BM and PB was 49% [13-98] and 30% [0-95], respectively. Inv(3)/t(3;3) was the only cytogenetic aberration in 10 patients (25%) and chromosome (chr) 7 abnormalities were the most common additional aberration, present in 22 patients (55%). CR after the first induction therapy was achieved in 9 patients (22%); 6 patients (15%) died during or shortly after induction therapy, and the remaining 25 (63%) were resistant. Salvage therapies for patients included re-induction with 7+3, FLAG-Ida, mitoxantrone-containing regimens, clofarabine-based protocols, or combination of hypomethylating agents with venetoclax. Seventeen (43%) patients underwent alloHSCT (10 in CR and 7 with active disease). When comparing intensive vs non-intensive induction regimen, no statistical significance was identified for complete remission (CR) rate (p= 1.00), overall survival (OS) rate (p=0.65) or relapse-free survival (RFS) rate (p=0.86). Similarly, no statistically significant prognostic factors (age, WBC, BM blast %, platelet count, hemoglobin, ANC, or time to transplant) for OS were identified. Interestingly, chr 7 abnormalities in inv(3) patients conferred a worse prognosis in this cohort (OS 41% vs. 59%, p= 0.0064, Figure 1B). This association was confirmed using a multiple Cox proportional hazards regression adjusting for age and blast percentage. Moreover, while chr 7 abnormality was also associated with a lower response rate (CR 14% vs. 40%), it did not reach statistical significance (p=0.12). Conclusions: In summary, de novo AML inv(3)/t(3;3) is a rare and aggressive subtype of AML conferring extremely poor prognosis. There appears to be an increase of incidence in females affecting a slightly younger demographic. Consistent with previous studies, the presence of chr 7 abnormalities was associated with even worse outcomes, which interestingly also drive EVI1 overexpression. The exact link between 3q26 loci rearrangements and chr 7 abnormalities warrants further investigation.

Abstract B003: EVI1 oncogenic role in non-canonical AR driven lethal prostate cancer

Abstract Androgen receptor (AR) remains active in castration-resistant prostate cancer (CRPC), and AR cistrome can become extensively reprogrammed with disease progression. Recent findings from our group and others have shown an association with non-canonical AR signature with resistance to enzalutamide. However, the complete mechanism is not fully revealed. EVI1 belongs to the zinc-finger transcription factor family, and overexpression of EVI1 is associated with poor patient outcomes. However, its role in the context of resistance to AR-targeted therapy remains unclear. Our preliminary data from ChIP-seq and GSEA analysis shows a significant enrichment of EVI1 target gene-set captured from annotated enzalutamide-resistant AR binding sites. Hypothesis: EVI1 cooperates in regulating the non-canonical AR activity associated with resistance to AR targeted therapy and blocking EVI1 may overcome resistance to target therapy. Methods : Using a PDX model of advanced enzalutamide-resistant CRPC, we performed single-cell ATAC-seq + gene expression multi-omic assay. Next, we determined the enrichment of canonical vs. non-canonical AR signatures across cell populations. Using motif analysis and transcription factor foot printing, we determine EVI1 (MECOM) motif z score and enrichment. Results: Our findings demonstrate enrichment of non-canonical AR signatures in distinct population of cells following exposure to enzalutamide. In addition, the subpopulation of cells enriched for non-canonical AR cistrome also exhibited high EVI1 (MECOM) motif z-scores. Conclusion : Our data suggests a potential link between EVI1 and non-canonical AR signaling. Ongoing mechanistic studies will provide insight mechanism by which EVI1 controls non canonical AR activity and its vulnerabilities. Citation Format: Surendra Gulla, Jonathan E. Bard, David J. VanderWeele, Kathleen Kelly, Remi Adelaiye-Ogala. EVI1 oncogenic role in non-canonical AR driven lethal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B003.

EVI1 exerts distinct roles in AML via ERG and cyclin D1 promoting a chemoresistant and immune-suppressive environment.

Aberrant expression of ecotropic viral integration site-1 (EVI1+) is associated with very poor outcomes in acute myeloid leukemia (AML), mechanisms of which are only partially understood. Using the green fluorescent protein reporter system to monitor EVI1 promoter activity, we demonstrated that Evi1high KMT2A-MLLT1-transformed AML cells possess distinct features from Evi1low cells: the potential for aggressive disease independent of stem cell activity and resistance to cytotoxic chemotherapy, along with the consistent gene expression profiles. RNA sequencing and chromatin immunoprecipitation sequencing in EVI1-transformed AML cells and normal hematopoietic cells combined with functional screening by cell proliferation-related short hairpin RNAs revealed that the erythroblast transformation-specific transcription factor ERG (E26 transformation-specific [ETS]-related gene) and cyclin D1 were downstream targets and therapeutic vulnerabilities of EVI1+ AML. Silencing Erg in murine EVI1+ AML models severely impaired cell proliferation, chemoresistance, and leukemogenic capacity. Cyclin D1 is also requisite for efficient EVI1-AML development, associated with gene expression profiles related to chemokine production and interferon signature, and T- and natural killer-cell exhaustion phenotype, depending on the interferon gamma (IFN-γ)/STAT1 pathway but not on CDK4/CDK6. Inhibiting the IFN-γ/STAT1 pathway alleviated immune exhaustion and impaired EVI1-AML development. Overexpression of EVI1 and cyclin D1 was associated with IFN-γ signature and increased expression of chemokines, with increased exhaustion molecules in T cells also in human AML data sets. These data collectively suggest that ERG and cyclin D1 play pivotal roles in the biology of EVI1+ AML, where ERG contributes to aggressive disease nature and chemoresistance, and cyclin D1 leads to IFN-γ signature and exhausted T-cell phenotypes, which could potentially be targeted.

Abstract 4900: Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression

Abstract EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc-finger transcription factor. Chromosome translocation t(3;3) or inv(3) at 3q26 repositions the enhancer (E) of the tumor suppressor GATA2 to induce EVI1 over-expression, while concomitantly repressing GATA2. EVI1 over-expression promotes self-renewal and blocks differentiation of leukemia stem-progenitor cells (LSCs) and causes therapy refractoriness and inferior overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor (BETi) is active against AML cells with inv(3)/t(3;3). BETi treatment also represses EVI1 and its targets c-Myc and Bcl-xL. We recently reported that tegavivint (TV) (BC-2059), a disruptor of the nuclear TBL1/R1-β-catenin-TCF7L2 complex represses c-Myc, cyclin D1 and Survivin and inhibits growth and survival of AML LSCs. In present studies, a CRISPR screen in UCSD-AML1 cells (with 3q26 lesion and EVI1 overexpression) with gRNAs targeting epigenetic regulators highlighted BRD4 and p300 as dependencies. Treatment with BETi, e.g., OTX015, dose-dependently induced apoptosis of AML cell lines and patient-derived (PD) AML cells (AML191 and AML194) with inv(3) or t(3;3). OTX015 treatment repressed c-Myc, c-Myb, CDK4, MECOM, and MCL1, while inducing HEXIM1 and cleaved PARP levels. TV treatment also dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions, associated with depletion of nuclear β-catenin, EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but increase in p21, CD11b, BIM and cleaved PARP levels. Hi-ChIP with H3K27Ac antibody demonstrated that TV treatment abolished chromatin loops between MYC and its Es within the super-E. RNA-Seq analysis showed negative enrichment (log2-fold) of gene sets of MYC, E2F and WNT targets, DNA replication/repair and reduction of the 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization of EVI1 and β-catenin with TBL1, also confirmed by the Proximity Ligation Assay. CyTOF analysis confirmed that TV treatment reduced EVI1, c-Myc, RUNX1, β-catenin, Bcl-xL, BCL2, MCL1 and Ki67 but increased cleaved PARP levels in the phenotypically characterized LSCs (with high expression of CLEC12A, CD123, CD244 and CD99) harboring inv(3) with EVI1 overexpression. Co-treatment with OTX015 and TV or the p300 inhibitor GNE-049 synergistically induced in vitro apoptosis in the AML cell lines and the PD AML cells with 3q26.2 lesions. Notably, in the flank-implanted PDX of AML191 or tail-vein infused PDX of AML242 models in NSG mice, treatment with OTX015 and TV, compared to vehicle control or each drug alone, significantly reduced AML growth and improved survival, without any host toxicity. These findings highlight the promising pre-clinical efficacy of novel BETi-based combinations against AML models harboring 3q26 lesions and EVI1 overexpression. Citation Format: Christine E. Birdwell, Warren C. Fiskus, Tapan M. Kadia, Christopher P. Mill, John A. Davis, Kaberi Das, Stephen Horrigan, Kapil N. Bhalla. Preclinical efficacy of targeting epigenetic mechanisms in AML with 3q26 lesions and EVI1 overexpression. [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 4900.

Abstract 3989: EVI1 oncogenic role in non-canonical AR driven lethal prostate cancer

Abstract Background: Androgen receptor (AR) remains active in castration-resistant prostate cancer (CRPC), and AR cistrome can become extensively reprogrammed with disease progression. Recent findings from our group and others have shown an association with non-canonical AR signature with resistance to enzalutamide. However, the complete mechanism is not fully revealed. EVI1 belongs to the zinc-finger transcription factor family, and overexpression of EVI1 is associated with poor patient outcomes. However, its role in the context of resistance to AR-targeted therapy remains unclear. Our preliminary data from ChIP-seq and GSEA analysis shows a significant enrichment of EVI1 target gene-set captured from annotated enzalutamide-resistant AR binding sites. Hypothesis: EVI1 cooperates in regulating the non-canonical AR activity associated with resistance to AR targeted therapy and blocking EVI1 may overcome resistance to target therapy. Methods: Using a PDX model of advanced enzalutamide-resistant CRPC, we performed single-cell ATAC-seq + gene expression multi-omic assay. Next, we determined the enrichment of canonical vs. non-canonical AR signatures across cell populations. Using motif analysis and transcription factor foot printing, we determine EVI1 (MECOM) motif z score and enrichment. Results: Our findings demonstrate enrichment of non-canonical AR signatures in distinct population of cells following exposure to enzalutamide. In addition, the subpopulation of cells enriched for non-canonical AR cistrome also exhibited high EVI1 (MECOM) motif z-scores. Conclusion: Our data suggests a potential link between EVI1 and non-canonical AR signaling. Ongoing mechanistic studies will provide insight mechanism by which EVI1 controls non canonical AR activity and its vulnerabilities. Citation Format: Surendra Gulla, Jonathan E. Bard, David J. VanderWeele, Kathleen Kelly, Remi Adelaiye-Ogala. EVI1 oncogenic role in non-canonical AR driven lethal prostate cancer. [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 3989.

Abstract 2122: The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML

Abstract The BRG1/BRM-associated factors (BAF) complex (also known as mSWI/SNF) is a critical regulator of the chromatin landscape of the genome. By controlling chromatin accessibility, BAF regulates lineage-specific transcriptional programs, including those important for AML blast cell growth and survival. FHD-286 is a highly potent inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4), which demonstrates strong tumor growth inhibition in several AML CDX and PDX models. FHD-286 is being developed for the treatment of relapsed/refractory AML and MDS (see Foghorn website for current status).Investigation of FHD-286 in a broad panel of AML cell lines showed that BAF inhibition affects hematopoietic transcriptional programs important for blast cell self-renewal and identity, agnostic of mutational background. Similarly, FHD-286 demonstrated broad efficacy in ex vivo treatment of AML patient-derived samples from diverse genetic backgrounds, including those with difficult to treat mutational profiles, including mtNPM1, FLT3 ITD, and Inv(3) with EVI1 overexpression. Interestingly, while higher concentrations (≥90 nM) of FHD-286 predominantly induced cytoreduction, lower concentrations (≤30 nM) predominantly led to differentiation-like responses. To investigate this differentiation effect, we performed immunophenotyping of cell lines and primary AML samples following prolonged treatment with FHD-286. Extended treatment (7+ days) with relevant concentrations (5-20 nM) of FHD-286 led to time- and dose-dependent upregulation of the myeloid differentiation marker CD11b, and acquisition of monocyte/metamyelocyte morphology. CD11b+ cells expressed lower levels of the hematopoietic transcription factor PU.1, as well as proliferation and survival proteins Ki67, Myc and BCL2. Additionally, primary patient samples showed a loss of CD34 positivity in blasts and GMP-like cells after 7 days of ex vivo treatment, indicating a decrease in stemness in these populations. Finally, upregulation of CD11b coincided with decreased BRG1 protein, suggesting that immature blasts are characterized by high levels of BRG1. These results suggest that BAF functions to drive transcriptional programs required to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by overcoming this differentiation block. Expanding on these findings, we have also demonstrated synergistic activity with multiple combination partners, including cytarabine and decitabine, in vitro. Elaboration of this in both CDX and PDX in vivo models also shows significant survival benefit in difficult to treat mutational backgrounds. Taken together, these findings suggest that FHD-286 is able to target blast progenitor populations that are heavily BRM/BRG1-dependent, and that combination with standard of care agents can achieve profound, mutationally agnostic antitumor activity in AML. Citation Format: Mike Collins, Astrid Thomsen, Ashley Gartin, Gabriel J. Sandoval, Ammar Adam, Sarah Reilly, Laure Delestre, Virginie Penard-Lacronique, Warren Fiskus, Kapil Bhalla, Stephane de Botton, Sam Agresta, Jessica Piel, Murphy Hentemann. The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML [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 2122.

The role of SF3B1 mutations in EVI1-rearranged myeloid neoplasms

In acute myeloid leukemia (AML), EVI1 rearrangement represented by inv(3)(q21q26) or t(3;3)(q21;q26) causes EVI1 overexpression via structural rearrangement of an enhancer, and confers poor prognosis. My colleagues and I performed a mutational analysis of EVI1-rearranged myeloid neoplasms and identified SF3B1, a core RNA splicing factor, as the most commonly co-mutated gene. Indeed, latent leukemia development in transgenic mice bearing the humanized inv(3)(q21q26) allele was significantly accelerated by co-occurrence of Sf3b1 mutation. Intriguingly, we found that this SF3B1 mutant induced mis-splicing of EVI1 itself, which generated an aberrant EVI1 isoform with in-frame insertion of 6 amino acids near the DNA-binding domain of EVI1. This aberrant EVI1 isoform exhibited DNA-binding activity different from wild-type EVI1 and significantly enhanced the self-renewal capacity of murine hematopoietic stem cells. We also identified the cryptic branch point and exonic splicing enhancer required for this EVI1 mis-splicing induced by the SF3B1 mutant. These data provide a basis for further elucidation of the molecular mechanism and potential therapeutic candidates for EVI1-rearranged AML.

Preclinical Efficacy of Novel Drug Combination Against AML with 3q26 Lesions and EVI1 Overexpression

The EVI1 gene is located at 3q26.2 in the MECOM locus and encodes for a zinc finger transcription factor (TF). EVI1 protein is expressed in leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. As a result of the chromosome translocation t(3;3) or inv(3) at 3q26, the distal enhancer of the TF and tumor suppressor GATA2 is dislocated and repositioned to induce expression of EVI1, while concomitantly repressing GATA2. EVI1 overexpression results in therapy refractoriness and inferior relapse-free and overall survival in AML. Previous reports highlighted that targeting BRD4 with BET inhibitor has activity against AML cells with inv(3)/t(3;3). Among the targets of EVI1 are the pro-growth and pro-survival proteins c-Myc and Bcl-xL, expressions of which are also regulated by enhancer activity that is undermined by BRD4 inhibition. We have recently reported that the nuclear TBL1/R1-β-catenin-TCF7L2 complex transcriptionally regulates c-Myc, cyclin D and Survivin and sustains AML stem cells, such that the undermining of this transcriptional axis by the TBL1/R1-β-catenin-binding disruptor tegavivint (BC-2059, Iterion) represses MYC, cyclin D1 and Survivin and inhibits growth and survival of AML stem-progenitor cells. Based on these observations, we interrogated the pre-clinical efficacy of pan-BET inhibitor OTX015 and/or tegavivint (TV) against AML cells with inv(3)/t(3;3) and overexpression of EVI1. Treatment with BET inhibitor OTX015 dose-dependently (100 to 1000 nM) induced apoptosis of AML cell lines (UCSD-AML1, OCI-AML20, MUTZ-3 and HNT-34) and patient-derived (PD) AML cells (AML191 and AML194) with inv(3)/t(3;3) and with or without monosomy 7. OTX015 treatment was associated with repression of c-Myc, c-Myb, CDK4, MECOM, and MCL1 as well as induction of HEXIM1 and cleaved PARP levels. We also determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines and PD AML cells with 3q26.2 lesions with/without monosomy 7. Furthermore, TV treatment resulted in attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, while inducing p21, CD11b, BIM and cleaved PARP levels. Next, utilizing H3K27Ac antibody Hi-ChIP (Arima Hi-ChIP), we interrogated the active gene regulatory interactions through a combined Hi-C and ChIP workflow, followed by Tn5 transposase-mediated on-bead library construction and NGS. We observed that in untreated AML191 cells numerous loop interactions were present between the super-enhancers and promoters respectively of MYC and BCL2. Following TV treatment (100 nM for 16 hours), differential loop calling revealed a loss of these interactions. RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of TGFβ, inflammatory response, TNFα and interferon signaling, and apoptosis signaling, but negative enrichment of gene sets of MYC, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in the phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99) harboring inv(3) and EVI1 overexpression. Co-treatment with TV and OTX015 or the BCL2 inhibitor venetoclax synergistically induced in vitro apoptosis (determined by SynergyFinder 2.0) in the AML cell lines and the PD AML cells. Notably, in the flank-implanted PD AML191 or tail vein AML242 models in NSG mice, treatment with TV and OTX015 for 2 to 5 weeks, compared to vehicle control or each drug alone, significantly reduced AML growth (p < 0.01). Furthermore, in the tail-vein infused and engrafted PD AML191 PDX model, treatment with TV and OTX015 or venetoclax for 6 weeks, as compared to vehicle control or each drug alone, significantly improved overall survival of the NSG mice (p < 0.01), without inducing any toxicity. These findings highlight the promising pre-clinical efficacy of novel combinations of TV and BET protein or venetoclax against AML models harboring 3q26 lesions and EVI1 overexpression.

Oncogene EVI1 Drives Acute Myeloid Leukemia Via a Targetable EVI1-CtBP1/2 Interaction and By Repressing Myeloid Regulator CEBPA

Overexpression of EVI1 (located at 3q26), as a result of 3q26 rearrangements, causes the development of one of the most aggressive, chemotherapy-resistant forms of AML. We hypothesize that insight into the function of EVI1 in AML will lead to the identification of targetable vulnerabilities for those patients. EVI1 encodes a DNA-binding zinc-finger protein that has a critical role in transcriptional regulation and maintenance of quiescent hematopoietic stem cells. Here, we aim to understand the mechanism by which EVI1 overexpression in bone marrow progenitors drives AML development. To identify its target genes, we made EVI1 rapidly degradable by endogenous fusion with an auxin-inducible degron (AID) tag in inv(3q21;3q26) rearranged MUTZ3 leukemia cells. Degradation of EVI1 strongly increased myeloid differentiation of MUTZ3 cells. Using metabolic labeling of newly produced RNA (SLAM-seq), we found 6 genes to be upregulated after 1.5 hrs of degradation. Out of those genes, CEBPA was also upregulated upon shRNA knockdown of EVI1 or after introducing inactivating mutations in the hijacked GATA2 enhancer that drives EVI1 expression. Those findings suggest persistent upregulation of CEBPA when EVI1 levels are reduced. In line with this, CEBPA expression is 1.83 fold (p=0.00024) lower in AML patients with 3q26 rearrangements (n=18) compared to non-3q26 rearranged cases (n=97). Interestingly, overexpression of CEBPA in MUTZ3 cells bypassed EVI1 driven repression and caused myeloid differentiation as well. We next addressed the question how EVI1 represses CEBPA transcription. The CEBPA locus contains 14 enhancers in a 100kb region around the gene body, among which the myeloid specific +42kb enhancer, essential for neutrophil development. EVI1 binds this +42kb enhancer of CEBPA as determined by chromatin immunoprecipitation sequencing (ChIP-seq) in 3q26-rearranged AML and MUTZ3 cells. After degradation of EVI1, H3K27Ac levels increased at this enhancer, indicating that EVI1 reduces activity of the +42kb CEBPA enhancer. Since EVI1 lacks any enzymatic domains that could modulate chromatin, we sought to further understand how EVI1 represses activity of the +42kb CEBPA enhancer. EVI1 protein immunoprecipitation followed by mass spectrometry revealed interaction with CtBP1/2, factors known to interact with repressors such as Polycomb proteins or HDACs. ChIP-seq showed a strong overlap between CtBP2 and EVI1 binding sites in MUTZ3 cells. Moreover, EVI1 elimination caused loss of CtBP2 binding to the chromatin including to the +42kb CEBPA enhancer. EVI1 harbors a PLDLS domain essential for binding to CtBP1/2. Mutation of this domain abolished CtBP2 binding and reduced transformation of mouse bone marrow progenitors by EVI1, as determined by in vitro replating assays. We reasoned we could exploit the high affinity of EVI1 for CtBP through this single domain by overexpressing a repeat of a small section of EVI1 containing only the CtBP-binding PLDLS-site. Overexpression of "4X-PLDLS competitor peptide" was able to sequester CtBP and thereby prevent it's binding to EVI1. The competitor peptide was able to abolish in vitro replating capacity and re-enabled differentiation of mouse bone marrow progenitors transformed with EVI1. In line with this, transduction of the competitor peptide into MUTZ3 cells led to upregulation of CEBPA as well as growth inhibition. Furthermore, in vivo leukemia outgrowth in a xenotransplant model of 3q26 rearranged AML cell lines MUTZ3 and SB1690CB was fully abolished by the competitor peptide, but not by a mutant control peptide. In conclusion, EVI1 is a repressor of active chromatin regions, including CEBPA via the +42kb myeloid specific enhancer. Repression by EVI1 is dependent on its interaction with CtBP1/2, proteins that recruit repressors of transcription via an essential . A PLDLS domain in EVI1 is essential for this interaction. The competitor peptide experiments point to exploiting this interaction with repressive molecules as a potential therapeutic target in AML with overexpression of EVI1.

Efficacy of Vcp/p97 Inhibitor, CB-5339, Alone and in Combinations Against High-Risk AML, Including Those with Genetic Lesion in TP53

Valosin-containing protein (VCP)/p97 or p97 is an AAA+ type ATPase involved in quality control of cellular proteins in normal and transformed cells, especially under cellular stress. VCP/p97 alleviates proteotoxic stress by facilitating bulk protein degradation through the ubiquitin-proteasome system (UPS), harnessing the energy of ATP hydrolysis to unfold and extract its ubiquitylated protein substrates from protein complexes or organelles and targeting them for proteasomal degradation, e.g., ER-associated degradation (ERAD). Targeted inhibition of p97 induces lethal ER stress. The vital role of p97 in protein homeostasis has been recognized as one of the key non-oncogenic addictions in transformed cells. Targeting p97 has been previously shown to yield preferential lethality in cancer versus normal cells and exhibit in vivo efficacy against xenograft models of cancers. By probing the CRISPR-screen dependency map (DepMap), we found that p97 is a dependency across AML cell lines, including those harboring alterations in genes that indicate an increased risk or poor prognosis. CB-5339 is a selective and potent, ATP-competitive inhibitor of p97, with only low inhibitory activity against other ATPases, AAA+ ATPases, and kinases. CB-5339 was previously shown to induce accumulation of polyubiquitylated proteins, retention of ERAD substrates, and lethal ER stress in cancer cells mediated by CHOP, DR5 and NOXA. In present studies, we determined that treatment with CB-5339 dose-dependently (200 to 1000 nM for 48 hours) induces in vitro loss of viability in AML cell lines (OCI-AML3, MOLM13, SET2 and HEL92.1.7 cells), as well as in fresh, patient-derived (PD) AML cells, including those with TP53 mutations and/or TP53 allelic loss, or with 3q26 (MECOM locus) lesions and EVI1 overexpression. Utilizing MOLM13 cells edited to express the missense mutation R175H or R248Q, or the null allele of TP53, introduced via CRISPR/Cas9 into the endogenous TP53 locus, we determined that CB-5339 was active against MOLM13 cells (with MLL-AF9, FLT3-ITD and wild-type TP53) and retained significant activity against MOLM13 cells with TP53-R175H, TP53-R248Q and MOLM13-TP53-/-. Comparing ATAC-Seq and RNA-Seq findings in CB-5339-treated versus -untreated MOLM13 cells we found concordant, significantly altered chromatin accessibility and mRNA expressions of gene-loci, e.g., increase in ATF4, ATF3, HSPA5 (GRP78), NOXA, BBC3 (PUMA), FBXO32, ITGAM, and CD86, but decrease in FOXA1, TLR4, CLEC12A, CXCR4 and IL7R. Following CB-5339 treatment, gene-set enrichment analyses (GSEA) showed significantly increased normalized enrichment scores of the gene-set involved in ER stress response, not only in MOLM13 with WT-TP53 but also in those with the TP53 mutations or allelic loss of TP53. However, treatment with CB-5339 increased levels of p-ATM, p-TP53 and TP53 only in MOLM13 and OCI-AML3 (each expressing only WT-TP53), without altering the protein levels of MDM2/4. In contrast, CB-5339-induced loss of viability correlated with augmented levels of ATF4, CHOP, as well as of pro-death proteins NOXA, DR5 and active BAX in AML cells regardless of TP53 status. Importantly, similar effects were also noted in PD AML cells with or without TP53 mutation. Co-treatment with relatively lower concentrations of CB-5339 (100 to 225 nM) and venetoclax (5 to 20 nM) exerted synergistic in vitro lethality in not only MOLM13 but also PD AML cells with WT-TP53, as well as in SET2 and PD AML cells harboring mutant TP53. Co-treatment with CB-5339 and pan-BET inhibitor OTX015 was also synergistically lethal against MOLM13 and PD AML cells with or without mutant TP53. Finally, in a tail-vein infused, luciferase transduced, aggressive xenograft model of MOLM13 cells, after AML engraftment, co-treatment for 3 weeks with CB-5339 (50 mg/kg/day, PO) and either venetoclax (30 mg/kg/day, PO) or OTX015 (30 mg/kg/day, PO), as compared to treatment with vehicle or each drug alone, significantly reduced the AML burden and improved median and overall survival of the NSG mice, without inducing significant toxicity. Taken together, these findings highlight that CB-5339 induces lethal ER stress in AML cells regardless of the TP53 status, and underscore the promise of CB-5339 treatment alone and in rational combinations in exerting efficacy against AML, including those with high-risk genetic alterations in TP53 or chromosome 3q26 lesions and EVI1 overexpression.

Abstract 2648: Novel combination therapies against AML with 3q26 lesions and EVI1 overexpression

Abstract The EVI1 gene is located on 3q26.2 and encodes a zinc fingers-containing transcription factor. Nuclear β-catenin-TBL1/R1-TCF7L2 axis activity and EVI1 expression characterize leukemia stem-progenitor cells, supporting their self-renewal and blocking differentiation. EVI1 is overexpressed in AML with chromosome translocation t(3;3) or inv(3) at 3q26, where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression confers poor response to therapy and inferior relapse-free and overall survival in AML. Tegavivint (BC-2059, Iterion) targets TBL1/R1 and disrupts its binding to β-catenin and TCF7L2, repressing MYC, cyclin D1 and Survivin, leading to apoptosis of AML cells. Here, we determined that treatment with TV (10-100 nM) dose-dependently induced apoptosis in AML cell lines (UCSD-AML1, MUTZ3 and OCI-AML20) and patient-derived (PD) AML cells (AML191 and AML194) with 3q26.2 lesions with/without monosomy 7. This was associated with attenuation of protein levels of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but upregulation of CD11b, BIM and cleaved PARP levels. Additionally, by reducing BRD4 occupancy at GATA2 enhancer and repressing EVI1, the pan-BET protein inhibitor OTX015 (100-1000 nM) also dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). RNA-Seq and gene set enrichment analysis in AML cells showed that TV treatment caused log2 fold-enrichment of gene sets of inflammatory response, TNFα and interferon signaling, TGFβ and apoptosis signaling, but negative enrichment of gene sets of c-Myc, E2F, DNA replication/repair, as well as showed reduction in expression of WNT targets and 17-gene stemness score. Confocal microscopy showed that TV treatment disrupted co-localization EVI1 and β-catenin with TBL1, also confirmed by Proximity Ligation Assay. Mass cytometry (CyTOF) analysis confirmed that TV treatment attenuated EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67 but augmented protein levels of APC and cleaved PARP in phenotypically characterized AML stem cells harboring t(3:3)/inv(3) and EVI1 overexpression (with high expression of CLEC12A, CD123, CD244, CD99). In vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (determined by SynergyFinder) of AML cell lines and the PD AML cells. In the tail-vein infused and engrafted PD AML191 cell model in NSG mice, treatment with TV and/or venetoclax or OTX015 for 6 weeks significantly reduced AML burden and improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. These findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 axis combined with BET protein or BCL2 inhibition is effective therapy against AML models harboring 3q26 lesions and EVI1 overexpression. Citation Format: Christine Birdwell, Warren C. Fiskus, Christopher P. Mill, John A. Davis, Qi Jin, Courtney D. DiNardo, Koichi Takahashi, Stephen Horrigan, Tapan M. Kadia, Naval Daver, Kapil N. Bhalla. Novel combination therapies against AML with 3q26 lesions and EVI1 overexpression [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 2648.

Resurgence of myeloproliferative neoplasm in patients in remission from blast transformation after treatment with hypomethylating agents

Subsequent blast (BP) or accelerated phase (AP) is a severe complication of Philadelphia-negative myeloproliferative neoplasms (MPNs). The prognosis is generally dismal, but hypomethylating agents (HMAs) may induce a long-lasting response in a minority of patients. Here, we report a cohort of six patients with BP/AP-MPN who experienced MPN relapse after a leukemia response was obtained with azacytidine. Five of the patients achieved complete remission despite the presence of characteristics associated with poor prognosis, such as complex and monosomal karyotypes, TP53 mutations, and EVI1 overexpression. These remissions persisted for over five years in four of the 6 patients. All patients showed rapid reemergence of MPN within a median of two months with thrombocytosis requiring the addition of anagrelide, hydroxyurea, or ruxolitinib given continuously in parallel with the azacytidine cycle. Serial JAK2 V617F allelic burden measurements showed little variation. Thromboembolic events occurred in 3 patients, one leading to death. These findings confirm that HMA may reverse the disease course in AP/BP-MPN to a more chronic phase that may last for years but also lead to morbidity and mortality. Combining maintenance therapy with HMA and MPN-specific drugs appears to be a possible approach to avoiding leukemia relapse and controlling MPN disease.

PRMT5 regulates ATF4 transcript splicing and oxidative stress response

Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.

Donor-Derived Leukemia in a Recipient of Double-Unit Cord Blood Transplantation for Acute Myeloid Leukemia: A Case Study and Literature Review.

We report a case of donor-derived leukemia (DDL) occurring 34 months after double-unit cord blood transplantation (CBT). Molecular analysis using short tandem repeat (STR) sequences proved the acute myeloid leukemia (AML) to be of dominant cord blood origin. Karyotype was normal and molecular analysis showed WT1 and EVI1 overexpression. Cytological and molecular remission were achieved with only induction and consolidation chemotherapy. Relapse occurred after 6 years of remission from one clone with only WT1 overexpression. Potential etiologies for donor cell leukemogenesis in the recipient are discussed, including occult leukemia in the donor or genetic predisposition to hematologic malignancies, impaired immune surveillance, induced or inherited stromal abnormalities, transformation of donor cells during engraftment via altered signals of the host tissues, and fusion of donor cells with residual leukemic cells leading to acquisition of oncogenes. Although cases of DDL occurring after umbilical CBT have already been reported, very few cases have been described arising after double-unit CBT. DDL cases following CBT previously described in the literature have been reviewed.

Pre-Clinical Efficacy of Targeting TBL1/R-β-Catenin-TCF7L2 Axis Against AML with 3q26 Lesions and EVI1 Overexpression

EVI1 gene maps to the MECOM locus at chromosome 3q26.2 and encodes for a zinc finger domain-containing transcriptional regulator. EVI1 supports hematopoietic stem cell self-renewal and blocks hematopoietic differentiation. EVI1 is overexpressed in up to 10% of AML, including those harboring chromosome translocation t(3;3)(q21;q26.2) or inv(3)(q21;q26.2), where the distal GATA2 hematopoietic enhancer is repositioned to induce EVI1 overexpression while repressing GATA2. EVI1 overexpression due to 3q26.2 lesions in MDS and AML is frequently associated with monosomy 7 and confers poor response to therapy and inferior relapse-free and overall survival. We had previously reported the pre-clinical efficacy of targeting TBL1/R1-nuclear β-catenin-TCF7L2 by tegavivint (BC-2059, Iterion Therapeutics) against AML stem/progenitor cells. This led us to interrogate the anti-AML activity of tegavivint (TV) in AML models harboring 3q26.2 lesions, where EVI1 overexpression has been documented to drive the biology of AML stem/progenitor cells. For this, we utilized AML cell lines with 3q26.2 lesions with/without monosomy 7 (UCSD-AML1, OCI-AML20, AML191 / MUTZ-3, AML194, HNT34), as well as patient-derived (PD) AML cells with 3q26 lesions with or without monosomy 7. Treatment with TV (10 to 100 nM) dose-dependently induced apoptosis in these cellular models. This was associated with attenuation of protein levels (determined by immunoblot analyses) of EVI1, TCF7L2, c-Myc, c-Myb, RUNX1, CEBPα, c-KIT, BCL2, Bcl-xL and MCL1, but upregulation of CD11b, BIM and cleaved PARP levels. Additionally, pan-BET protein inhibitor OTX015 (100 to 1000 nM) dose-dependently induced apoptosis of AML cell lines and PD AML cells with t(3:3)/inv(3). Following TV treatment, RNA-Seq and gene set enrichment analysis in UCSD-AML1 and OCI-AML20 cells showed log2 fold-changes in gene expression and positive enrichment of pathway genes and/or reactomes of inflammatory response, TNFα and interferon signaling, TGFβ, NOTCH and apoptosis signaling, as well as negative enrichment of gene sets of c-Myc, E2F, G2M checkpoint, DNA replication and repair and chromosome maintenance (all with FDR q-values < 0.1). QPCR analysis showed repression of EVI1, MYC and KIT, but upregulation of Axin2 mRNAs. Following TV treatment, confocal microscopy showed reduction of nuclear protein levels of EVI1 and β-catenin, as well as disrupted their co-localization with TBL1. Proximity ligation assay also demonstrated that exposure to TV significantly reduced the proximity of TBL1 and EVI1 as well as of TBL1 and β-catenin. Mass cytometry (CyTOF) analysis of patient-derived (PD) AML samples with t(3:3)/inv(3) confirmed that TV treatment attenuated protein levels of EVI1, c-Myc, RUNX1, β-catenin, TBL1/R1, Bcl-xL, BCL2, MCL1 and Ki67, but augmented protein levels of APC and cleaved PARP in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). Consistent with effects of TV on gene-expressions that regulate cell death pathways, in vitro co-treatment with TV and BCL2 inhibitor venetoclax or OTX015 synergistically induced apoptosis (as determined by the SynergyFinder algorithm) of AML cell lines and PD AML cells with t(3:3)/inv(3) and EVI1 overexpression. In the in vivo HNT-34 model of flank-implanted and established tumors in athymic nude mice, treatment with TV (50 mg/kg, TIW, IP) and venetoclax (30 mg/kg, PO, daily) for 5-weeks yielded more tumor growth delay than vehicle control or TV alone. Additionally, following tail vein infusion and engraftment of luciferized AML191 cells in NSG mice, treatment with TV and/or venetoclax or OTX015 (30 mg/kg, PO, daily) was evaluated for 6 weeks. Co-treatment with TV and venetoclax or TV and OTX015 significantly reduced AML growth (determined by reduction in bioluminescence by Xenogen camera) (p < 0.05), as well as improved overall survival of the NSG mice more than treatment with each drug alone or vehicle control, without any toxicity. Collectively, these findings highlight that targeted inhibition of TBL1/R1-nuclear β-catenin-TCF7L2 by treatment with TV also inhibits EVI1 and its targets. They also demonstrate superior pre-clinical efficacy of novel TV-based combinations with BCL2 or BET protein inhibitor against AML models harboring 3q26 lesion and EVI1 overexpression. DisclosuresDiNardo: Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; AbbVie: Consultancy, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding. Takahashi: Symbio Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Novartis: Consultancy; Celgene/BMS: Consultancy. Horrigan: Iterion Therapeutics: Current Employment. Kadia: Astellas: Other; Cure: Speakers Bureau; Genfleet: Other; Ascentage: Other; Jazz: Consultancy; Sanofi-Aventis: Consultancy; Dalichi Sankyo: Consultancy; Cellonkos: Other; Novartis: Consultancy; AstraZeneca: Other; BMS: Other: Grant/research support; Liberum: Consultancy; Amgen: Other: Grant/research support; Pulmotech: Other; Pfizer: Consultancy, Other; Genentech: Consultancy, Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support.