Primary Patient Samples Research Articles

Intracellular IL-23 Receptor (IL-23R) Is Necessary for AML Viability and Regulates Mitotic Spindle and Centrosome Formation

The IL-23 receptor (IL-23R) is a cell surface receptor classically expressed on T cells. In response to inflammatory stimuli, such as microbial infection, dendritic cells and macrophages secrete the cytokine IL-23, which stimulates IL-23R leading to increased T cell activation and proliferation. Here, we identify a novel function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation. An unbiased in silico screen was performed using the Interlinked Therapeutics ITX machine learning platform to identify novel targets and mechanisms in AML. This analysis identified IL-23R as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed IL-23R protein was over-expressed >2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression (n=3) in normal hematopoietic cells. IL-23R is classically a cell surface receptor, and we confirmed cell surface localization in DNT (Double Negative T) cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly in the cytoplasm and nucleus as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To validate that we were indeed detecting IL-23R, we confirmed intracellular localization in AML cells using 4 different antibodies directed against 4 different epitopes of the receptor. To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to the BioID controls, we identified 61 proteins that preferentially interacted with IL-23R. Of the 61 proteins, 36 are known cytoplasmic or nuclear proteins. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using a Proximity Ligation Assay and confocal microscopy, we validated the interaction of endogenous IL-23R with the known mitotic spindle proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells. In addition, by confocal microscopy, we demonstrated that IL-23R co-localizes with the mitotic spindle and centrosomes in AML. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. We then evaluated the effects of IL-23R knockdown on AML growth and viability. Knockdown or knockout of IL-23R reduced AML growth in OCI-AML2, TEX, K562, NB4, and U937 cells. In addition, knockdown of IL-23R led to a significant reduction in engraftment efficiency of TEX cells into murine bone marrow. To evaluate the effects of IL-23R depletion on normal hematopoiesis, we evaluated constitutive homozygote IL-23R knockout mice. IL23R-/- mice were bred in normal ratios and grew similar to wild type mice. IL-23R was associated with the mitotic spindle and centrosomes in hematopoietic cells of wild type mice and no IL-23R was detected in the knockout mice. Complete blood counts did not differ compared to wild type mice. The abundance of HSC (hematopoietic stem cell) and LSK (Lin−Sca-1+c-Kit+) hematopoietic stem cells were also similar between IL-23R -/- and wild type mice. In summary, we discovered that IL-23R is necessary for AML growth and viability by regulating the formation of the mitotic spindle and centrosome. Thus, we identified a new function and localization for IL-23R and highlight a new therapeutic target for this disease.

The Composition of Acute Myeloid Leukemia Cell Differentiation States Predicts Response to Immune Checkpoint Blockade

Background: Given the activity of allogeneic T cells in acute myeloid leukemia (AML) patients, immunotherapies stimulating antitumor T cell responses have been explored as a promising therapeutic option. However, these therapies have been associated with modest clinical activity, and further research is needed to identify response and resistance mechanisms. A spectrum of primitive and differentiated cells comprise AML in each individual patient, and this cell composition has been previously shown to predict outcomes. In particular, high proportions of AML stem and progenitor cells have been associated with worse clinical outcomes and lack of response to induction chemotherapy (Ng et al Nature 2016; van Galen et al Cell 2019; Zeng et al Nat Med 2022). The composition of AML cell differentiation states in relation to immunotherapy response or resistance has not been elucidated; herein, we explored whether this composition may be associated with response in AML patients treated with immune checkpoint blockade. Methods: Pre- and posttreatment whole bone marrow (BM) samples were collected from 31 newly diagnosed and relapsed/refractory AML patients treated with azacitidine and pembrolizumab (NCT02845297) and 18 relapsed/refractory AML patients treated with high-dose cytarabine (HiDAC) and pembrolizumab (NCT02768792; Zeidner et al Blood Cancer Discov 2022) on two separate phase II trials. Primary patient samples and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki as per research protocols approved by the Institutional Review Boards of the participating institutions. Bulk RNA sequencing was performed on all BM samples. Differential gene expression analysis was performed using the DESeq2 package, and gene set enrichment analysis (GSEA) was performed using the clusterProfiler package. Proportions of AML cell populations present in BM samples were deconvoluted using CIBERSORTx as per Zeng et al Nat Med 2022. The LSC17 score and LinClass7 score were calculated as per Ng et al Nature 2016 and Zeng et al Nat Med 2022, respectively. All analyses and graphs were generated using R (v4.2.1). Results: GSEA using gene sets from a range of primitive and differentiated AML and normal cell types as per van Galen et al Cell 2019 was performed on pre- and posttreatment samples. Patients achieving a complete response (CR) to either immunotherapy regimen were enriched at baseline for the following differentiated AML gene sets: pro monocyte (Promono)-like AML; monocyte-like AML; conventional dendritic cell (cDC)-like AML; and myeloid-like AML (Figure 1A). Patients not responding (NR) to azacitidine/pembrolizumab were enriched for the following stem and progenitor AML gene sets: hematopoietic stem cell (HSC)-like AML; progenitor (Prog)-like AML; and HSC progenitor (HSCProg)-like AML (Figure 1A). To validate these findings, BM samples were deconvoluted into inferred fractions of AML differentiation states using CIBERSORTx and single-cell RNA sequencing expression data from Zeng et al Nat Med 2022. Patients not responding to azacitidine/pembrolizumab had a higher proportion of leukemia stem and progenitor quiescent cells (LSPC Quiescent) at baseline, while patients achieving CR to either regimen had higher baseline proportions of pro monocyte-like AML cells (Figure 1B). Additionally, patients not responding to azacitidine/pembrolizumab had higher baseline LSC17 and LinClass7 scores, two gene expression scores that are reflective of leukemia stemness (Figure 1B). In the posttreatment setting for both regimens, HSC-like, Prog-like, and HSCProg-like gene sets were associated with NR; monocyte-like and myeloid-like gene sets were associated with CR (Figure 1A). Discussion: Patients achieving CR to azacitidine/pembrolizumab and HiDAC/pembrolizumab had higher proportions of differentiated AML cells, while NR patients had a higher proportion of AML stem and progenitor cells. We hypothesize that antitumor T cells may preferentially target differentiated AML cells over AML stem and progenitor cells. Further research is needed to validate these findings in a prospective and mechanistic manner and in other immunotherapy regimens. Analyses of the proportions of AML differentiation states in patients relapsing after allogeneic hematopoietic cell transplantation are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting HIF-Hydroxylases Compromises Disease Initiation and Propagation, and Synergises with Current Therapies to Eliminate Cancer Stem Cells in AML

Acute myeloid leukaemia (AML) is an aggressive disorder of haematopoietic stem and progenitor cells (HSPCs), in which they acquire mutations, resulting in generation of disease fuelling, and ultimately treatment-resistant, leukaemic stem cells (LSCs). Given the hypoxic nature of the bone marrow microenvironment, the role, and pharmacological manipulation of the Hypoxia Inducible Factor (HIF) pathway in normal and malignant haematopoiesis is an area of significant interest. We, and others, previously found that HIF-α, the master regulator of the pathway, is not essential for normal haematopoiesis and its deletion promotes leukaemogenesis. Here we reveal through genetic, pharmacological and omics-based investigations that HIF stabilisation, through inhibition of its regulator PHD2, is a novel potent therapeutic strategy in AML. First, to genetically dissect to role of PHD2 in the initiation of AML, we transformed Phd2cKO HSPCs with AML-associated oncogenes and found that AML cells lacking Phd2 were compromised in vitro, established a weaker leukemic burden and initiated AML with a significantly longer latency in vivo. Moreover, employing our iMLL-AF9;shPhd2 mice, which upon DOX activate MLL-AF9 and a short-hairpin targeting Phd2, we found that Phd2 knockdown in vivo significantly delayed the onset of AML. Next, to investigate acute Phd2 depletion in newly diagnosed disease, we transplanted transformed shPhd2 cells, and upon AML detection induced expression of shPhd2. Notably, Phd2 knockdown in established AML cells compromised disease progression and significantly improved survival of leukaemic mice. Following this, we examined the impact of Phd2 inactivation on HSC function and multilineage haematopoiesis. Phd2cKO mice displayed no adverse phenotypes, and Phd2-deficient HSCs were able to successfully sustain multilineage haematopoiesis upon transplantation. Moreover, global DOX-inducible Phd2 knockdown had little effect on haematopoiesis. Thus, PHD2 inactivation compromises AML development and maintenance, but does not impact normal haematopoiesis, suggesting an ample therapeutic window for its inhibition. Given our genetic evidence that PHD2 is a promising target in AML, we explored the potential of its inhibition. We employed a commercially available clinical trial grade PHD inhibitor, as well as our own novel, selective, and potent PHD inhibitor, both of which work through distinct mechanisms. Significantly, both inhibitors compromised the growth and viability of murine AML cells, as well as multiple established human AML cell-lines and primary patient samples with diverse mutational backgrounds. Moreover, we have also discovered that PHD inhibition markedly synergises with AML treatments currently used in the clinic, thus enhancing their anti-leukaemic effect. Investigating the molecular mechanisms behind our findings, we found HIF-α stabilisation across multiple AML human cell lines following PHD inhibition. Furthermore, we utilised our HifαcKO leukaemic cells which, unlike control cells, were not affected by PHD inhibition, indicating that the anti-leukaemic effect of PHD inhibition is indeed HIF-dependent. Next, we performed RNA-seq and proteomic analyses following genetic and pharmacological PHD inhibition. These experiments revealed increased expression of key myeloid differentiation genes, glycolytic pathway, and a consistent upregulation of HIF target genes, including the pro-apoptotic BNIP3. Finally, pharmacological stimulation of apoptosis combined with PHD inhibition displayed a further and profound anti-leukaemic effect. Taken together, our data strongly indicate that genetic deletion and specific potent pharmacological inhibition of PHD2 can upregulate HIF-dependent pathways, eradicating AML disease in vitro and in vivo, thus unveiling a novel promising therapeutic target.

Electron Transport Chain Complex V Inhibition Enhances the Antileukemic Activity of Low-Dose Venetoclax in Acute Myeloid Leukemia

Background: BCL-2 dependent leukemia-stem cells preferentially rely on oxidative phosphorylation (OXPHOS) as a source of energy (Pei et al Cancer Discovery 2020; Stevens et al Nature Cancer 2021). BCL-2 inhibition through the use of venetoclax (VEN) has been linked to disruption of OXPHOS via reduced complex II activity. Given the use of OXPHOS by BCL-2 dependent AML cells, we sought to potentiate VEN effects by further perturbing electron transport in AML. Recently, we discovered that Ammocidin, a glycomacrolide natural product, targets leukemia OXPHOS via inhibition of ATP synthase, complex V of the electron transport chain (Reisman et al Nature Chem Bio 2022). Here, we explored the therapeutic window of simultaneous inhibition of Complex V and BCL-2 by ammocidin and VEN, respectively, in AML cells. Methods: We treated AML cell lines MV-4-11 and MOLM-13 with ammocidin and evaluated for viability via Cell Titer-Glo. To validate the combination of ammocidin and VEN, in vivo, we transplanted the MV-4-11 cell line into immunocompromised NSGS mice. Engrafted mice were treated with either standard of care (SOC) VEN (25 mg/kg 5x/week) + azacitidine (AZA) (0.2mg/ml 3x/week), ammocidin (0.1 mg/kg 5x/week) monotherapy, or a combination of each at these doses. Total human CD45+/CD33+ cells were removed from marrow, spleen and peripheral blood of xenografted mice and measured via flow cytometry. To further discover the efficacy of ammocidin/VEN in heterogenous AML samples, we treated primary patient AML cells with VEN, ammocidin or combination therapy. Mononuclear bone marrow cells from nine mutationally diverse patients with AML were tested and evaluated for cell viability via Cell Titer-Glo. To verify induction of apoptosis, we subjected cultured patient samples to annexin V/PI staining 24 hours after exposure to 300nM ammocidin and 100nM VEN single and combination therapy. Results: Cell Titer-Glo assays revealed that AML cell lines MV-4-11 and MOLM-13 cells were sensitive to complex V inhibition with a GI50 of 45 and 131nM. Combining ammocidin with VEN was synergistic in both cell lines. In vivo, the addition of ammocidin to an SOC regimen revealed significant decreases of AML cells in the bone marrow (SOC vs/ SOC+ ammocidin p=0.004), blood (SOC vs/ SOC+ ammocidin p=0.04) and spleen (SOC vs/ SOC+ ammocidin p=0.006). When looking at primary AML samples, combination therapy was more effective than single-agent therapy (Ven vs VEN+ ammocidin p=0.02). Induction of apoptosis, noted through Annexin+/PI− cell staining, was significantly enhanced with the combination therapy in AML blast cells at 24 hours (VEN vs VEN+ ammocidin, p=0.007). Further studies are underway to test this combination in both VEN sensitive and resistant AML PDX primagraft models in vivo. Conclusion: Targeting complex V and BCL-2 together resulted in the decreased expansion of human AML cell lines, and primary patient samples; and there was a preferential reduction of AML in vivo. These preliminary data represent a potential for ammocidin/VEN therapy in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of ERK1/2 Reverses Venetoclax-Induced Autophagy to Overcome Resistance in Acute Myeloid Leukemia

Background: Activation of the RAS/MAPK pathway confers inherent/acquired resistance to the Bcl-2 inhibitor, venetoclax, in AML (Zhang et al., Signal Transduct Target Ther. 2022). Pro-survival Bcl-2 inhibits apoptosis as well as autophagy. Phosphorylation of Bcl-2 by ERK1/2 leads to dissociation of the autophagic protein Beclin 1 from Bcl-2 and promotes autophagy (Pattingre et al., Cell. 2005). Thus, while Bcl-2 inhibition can induce apoptosis, its inhibition leads to induction of autophagy (Pattingre et al., Cell. 2005) that can protect tumor cells from organelle damage and nutrient deprivation. Objective: We hypothesized that terminal MAPK pathway inhibition using an ERK1/2 inhibitor would reverse venetoclax-induced autophagy and synergistically lead to cell death/apoptosis. Methods: We inhibited ERK1/2 using Compound 27 (Heightman et al., J Med Chem. 2018), a close analog of ASTX029, which is a dual-mechanism ERK1/2 inhibitor currently under clinical investigation in solid tumors (NCT03520075). We studied the potential synergy of ERK1/2 and Bcl-2 inhibition in decreasing cell proliferation and inducing apoptosis and in overcoming venetoclax resistance in AML preclinical models and primary patient samples (n=8). We performed simultaneous analysis of cell proliferation, stress response and DNA damage using single cell proteomics analysis to understand the mechanism of synergy. Autophagy was measured by lipidated LC3 quantification (flow cytometry and immunoblotting). Colony formation assay using NRAS-mutant PDX was performed to analyze the effect on progenitor cells. For insights into the mechanisms of synergy, we conducted large-scale proteomics analyses by single cell mass cytometry (CyTOF) and simultaneously measured cell proliferation, stress response, DNA damage and Bcl-2 family proteins. Results: Inhibition of ERK1/2 sensitized OCI-AML3 (NRAS-mutant) cells (Combination Index [CI]: 0.008) and venetoclax-resistant OCI-AML2 cells (CI: 0.05) to venetoclax and shifted the cytostatic effect of the single agents to a cytotoxic effect. Combination treatment induced apoptosis in leukemia initiating progenitor cells (LICs, CD34+CD38-) in primary AML samples with 54% apoptosis in combination versus 10% with venetoclax alone (CI = 0.03-0.23), while sparing normal CD34+CD38- cells. Mechanistically, basal ERK1/2 phosphorylation and autophagy (LC3 lipidation), were higher in venetoclax-resistant OCI-AML2 cells versus parental cells (p=0.0008) and both were inhibited with ERK1/2 inhibition alone and in combination with venetoclax. Simultaneous analysis of cell proliferation, stress response and DNA damage showed downregulation of proliferation (Ki-67) and an increase in ATF4 in response to ERK1/2 inhibition. The combination of Compound 27 and venetoclax inhibited autophagy (lipidated LC3) and increased apoptosis (increased cleaved PARP and cleaved caspase 3, Fig 1a). Venetoclax-induced autophagy was also confirmed in OCI-AML3 cells at 24 h post treatment (p=0.03). ERK1/2 inhibition reversed venetoclax-induced autophagy in OCI-AML3 (p=0.0003) and OCI-AML2-venetoclax-resistant cells (p=0.0007). CyTOF analysis showed decreased p-AMPK in response to ERK1/2 inhibition in OCI-AML3 cells. As AMPK/FIS1-mediated mitochondrial autophagy is required for self-renewal of AML stem cells (Pie et al., Cell Stem Cell. 2018), we confirmed decreased clonogenic potential of AML progenitor cells by reduced colony formation in response to ERK1/2 inhibition using an NRAS-mutant AML PDX model (p=0.0002). Single cell proteomics (CyTOF) showed decreased p-AMPK in response to single agent ERK1/2 inhibition or the combination with venetoclax in LIC populations (CD34+CD38-) in the NRAS-mutant AML PDX model. In addition to p-AMPK the expression of cMyc and CD44 was also reduced in the combination treatment, indicating reduced stemness (Fig 1b). Conclusion: Mechanistically, our results indicate that pharmacological inhibition of ERK1/2 impairs autophagy and sensitizes venetoclax-resistant AML cells to apoptosis, thus overcoming primary and secondary resistance to venetoclax. In addition, combination treatment induced apoptosis of LICs and reduced clonogenic potential and markers of stem cell function. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Combination of Clinical Menin Inhibitor Ziftomenib and the Nuclear Export Inhibitor Selinexor Synergistically Inhibit MLL-r AML

PURPOSE: Menin is a scaffold protein that tethers histone-lysine-N-methyltransferase MLL1 (KMT2A) to chromatin by binding to the menin binding domain (MBD) of KMT2A. The N-terminus of the KMT2A gene recombines with a range of genes to form more than 80 fusion proteins (FP) in MLL-rearranged (MLL-r) AML. In myeloid stem progenitor cells, the menin-KMT2A complex modulates the expression of the leukemogenic homeobox A9 (HOXA9) gene, as well as its co-factor, MEIS1. Menin inhibitors evict menin from the chromatin and reduce KMT2A and KMT2A-FP binding to their targets in MLL-r leukemia, breaking the differentiation block and inducing tumor regression. Aberrant nuclear export is common in cancer, resulting in anomalous localization of various proteins, including tumor suppressor proteins (TSPs). Blocking XPO1/CRM1 using a selective inhibitor of nuclear export (SINE) compound selinexor has been shown to have robust anti-leukemia activity. In the present study, we hypothesized that inhibition of menin and nuclear export would synergistically suppress AML cell proliferation and survival. We have used the clinical-stage menin inhibitor ziftomenib and selinexor to simultaneously target menin-KMT2A protein-protein interactions and nuclear export. METHODS: To assess growth inhibition, an ATP-based cell proliferation assay was used. Combination synergy was determined using CalcuSyn Version 2.0 synergy software. Stem-like progenitor cells were isolated using the StemSpan CD34+ expansion kit (StemCell Tech). Colony formation efficiency was determined using a Methocult assay (StemCell Tech). Gene and protein expression and cell death were detected using quantitative real-time PCR, western blotting, and annexin-V/PI fluorescence-activated cell sorting, respectively. RESULTS: The clinical candidate ziftomenib, in combination with selinexor, synergistically inhibited the growth of MLL-r AML cell lines (MV4;11, MOLM13, and SEM) (CI<1) [Fig.1A]. The combination of menin and XPO1 inhibitors significantly suppressed colony formation of CD34+ MLL-r progenitor cells derived from primary patient samples but was not toxic to normal CD34+ human hematopoietic progenitor stem cells (HPSCs). Menin inhibitor treatment caused the downregulation of menin protein along with its downstream targets HOXA9, MEIS1, and FLT3 in western blot analysis. Interestingly, the nuclear export inhibitor selinexor also reduced menin, HOXA9, and MEIS1 protein levels. The combination of menin and XPO1 inhibitors enhanced menin down expression, suggesting a molecular basis for the synergy between the two compounds [Fig 1.B]. Ziftomenib suppressed the expression of menin in a time-dependent manner, which is evident as early as six hours of treatment. This combination significantly enhanced both early and late apoptotic cell death in MLL-rearranged AML cells compared to either compound alone. We have performed total RNA sequencing and observed differential expression of certain mRNAs and micro RNAs (miRs). The TMEM191B, SH3TC1, and SULT1C2 mRNAs were upregulated by KO539 and Selinexor, which are enhanced further by combination treatment. On the contrary, CENPK, CKS2, KIF18A, and H2BC4 mRNAs were downregulated by a single agent or combination. Among miRs, hsa-miR-34a-5p, hsa-miR-142-3p, hsa-miR-26b-5p were upregulated and hsa-miR-33b-3p, hsa-miR-4454, hsa-miR-210-3p and hsa-miR-130b-3p [reported to be an essential lineage-specific codriver of MLL-AF4 leukemia] were downregulated by single agent or combination. Such mRNAs and miRs panels could serve as biomarkers for treatment response. CONCLUSION: These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and nuclear export could be a viable strategy for the treatment of MLL-r AML. Ongoing experiments with CDX and PDX using MLL-r models and functional analysis using total transcriptome sequencing and proteomics will be presented at the ASH meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Exportin, XPO2, Is a Novel Therapeutic Target in Pediatric, Adolescent and Young Adult AML

The active transport of proteins across the nuclear membrane is regulated by nuclear transport receptors termed importins and exportins, depending on their direction of transport. Nuclear transport receptors contribute to cancer development, through abnormal localization of protein cargos. Increased expression of the exportin, exportin-1/chromosome region maintenance 1 (XPO1/CRM1), has been associated with high risk AML and shorter survival times and XPO1 inhibitors show signs of efficacy in AML. While previous data demonstrate a functional role for XPO1 in AML, less is known about the exportin, XPO2, (alias CSE1L). Despite XPO2 RNAi depletion screens demonstrating a higher genetic dependence in leukemia versus solid tumors/non-hematologic malignancies (p = 0.0394), the functional impact of inhibiting XPO2 has never been studied in leukemia. We analyzed a public database of CRISPR/Cas9 depletion screens in 26 AML cell lines. XPO2 ranked in the top 1.4% of essential genes in AML, along with KPNB1 and XPO1 (DepMap 22Q2 Public+Score, Chronos, www.depmap.org). An RNAi depletion screen confirmed these findings, with XPO2 identified within the top 0.2% of essential genes in AML (Achilles+DRIVE+Marcotte, DEMETER2, www.depmap.org). Gene dependency scores indicated a greater reliance on XPO2, compared to XPO1 (p <0.0001). Increased expression of XPO2 mRNA was associated with decreased event free survival (p = 0.0014) and overall survival (p = 0.00091) in pediatric and adolescent/young adult (AYA) AML patients from the TARGET dataset. Analyzing the BeatAML dataset, increased XPO2 mRNA expression was also associated with decreased overall survival in AML patients <40 years, but not in patients >40 years (p <0.0001). We analyzed protein expression databases and identified XPO2 as the highest expressed member of the exportin family in AML cells lines and AML primary patient samples. By immunoblot analysis, increased expression of XPO2 was detected in 7/7 primary AML samples and 8 AML cell lines (HL60, MV4-11, MOML13, NB4, OCI-AML2, OCI-M2, TEX and U937), compared to normal hematopoietic cells. XPO2 mRNA was also increased in 5/5 primary AML samples and 8/8 AML cell lines, compared to normal hematopoietic cells, as measured by qRT-PCR. To confirm the functional importance of XPO2 in AML, we knocked down XPO2 with shRNA in AML cell lines. Genetic silencing of XPO2 decreased growth and viability of OCI-AML2, TEX and NB4 cells. Gambogic acid (GA), a natural product from Garcinia hanburyi, has been identified as a covalent inhibitor of XPO2-mediated nuclear transportation. In OCI-AML2, TEX and NB4 cell lines, gambogic acid decreased growth and viability of AML cell lines at nanomolar concentrations (TEX cells IC50 = 369nM, OCI-AML2 cells IC50 = 433nM, NB4 cells IC50 = 161nM). To uncover the mechanism of how XPO2 mediates cell death in AML we interrogated a publicly available BioID database (PXD007976) and performed gene set enrichment analysis on XPO2 targets using gprofiler. Processes related to ribosome biogenesis were significantly enriched in XPO2 cargo, whereas XPO1 cargo were enriched for processes related to snRNA transcription and transport (p <0.05). In summary, we identified XPO2 as an essential gene for AML. XPO2 is preferentially increased in AML cell lines and primary patient samples compared to normal hematopoietic cells. Increased XPO2 expression is associated with worse clinical outcomes in pediatric and AYA patients with AML. XPO2 may function to enhance ribosomal biogenesis, demonstrating a critical role in leukemia initiation and progression. Thus, this work demonstrates a new dependency of AML on XPO2, as well as highlights a new therapeutic target for this disease.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Altering Drp1-Dependent Mitochondrial Fission and Metabolism

Background: Mutational (RAS, PTPN11 etc.) or non-mutational activation of RAS/RAF/ERK pathway is associated with primary or secondary resistance to venetoclax in AML therapy (Zhang et al., Sig Transduct Target Ther. 2022). While upregulation of alternate antiapoptotic proteins e.g. Mcl-1 has been implicated in resistance to venetoclax (Zhang et al., Sig Transduct Target Ther. 2022) role of mitochondrial dynamics (fission/fusion) in venetoclax resistance has not been well explored. Oncogenic RAS activates downstream ERK1/2 phosphorylation and mitochondrial fission through ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1 (Nagdas et al., Cell Rep. 2019). Drp1 deletion inhibits tumorigenesis of KRAS driven pancreatic cancer, but the importance of RAS driven changes in mitochondrial dynamics in AML is poorly defined. ERK1/2 inhibition may therefore provide insights into the dependence of AML cells on altered mitochondrial dynamics and metabolism driven by oncogenic RAS. Objectives: We assessed the synergistic anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and in primary patient samples (n=8). Further, we investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., J Med Chem. 2018) a close analog of ASTX029, which is currently under clinical investigation in solid tumors (NCT03520075), was highly synergistic with venetoclax at inducing apoptosis in AML cells. NRAS-mutant OCI-AML3 and THP1 cells, that are intrinsically resistant to venetoclax, were sensitized to venetoclax by ERK1/2 inhibition with a combination index (CI) of 0.008 (cell death ~90% for combination versus 20% with venetoclax alone). Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax alone (CI=0.6). The combination was also effective in eliminating bulk and CD34+CD38- leukemia initiating cells (LICs) in primary AML samples (CI:0.03-0.23). Colony formation assay and single cell level CyTOF analysis using an NRAS-mutant PDX showed markedly impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to single agent ERK1/2 inhibition and combination treatment, indicating a potential impact on stemness. Mitochondrial reactive oxygen species (ROS) mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML (Schimmer et al., Cell Stem Cell. 2018). As a result of ERK1/2 inhibition, pDrp1 (Ser616) levels decreased, and mitochondrial length (p<0.001) and mitochondrial ROS levels (p<0.01) were increased, suggesting impaired fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Rescue with overexpression of active phospho-mimetic form of Drp1 i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells resulted in decreased apoptosis and reversed the synergy between ERK1/2 and Bcl-2 inhibition when compared to wild type and phospho-null (Ser616Ala) Drp1 overexpression (p<0.001, Fig 1a). Of note, overexpression of Drp1-Ser616Glu-Ser637Ala phospho-mimetic led to decreased mitochondrial length (Fig 1b), suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, decreased maximal oxygen consumption rate (p<0.05) and ATP production (p<0.05), as well as reduced ROS production (p<0.01). Moreover, combination treatment failed to significantly induce ROS production in cells expressing the phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Collectively, these results indicate that inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. Conclusion: ERK1/2 inhibition alters Drp1 dependent mitochondrial dynamics and metabolism, resulting in mitochondrial defects and overcoming resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Menin Inhibitor Ziftomenib (KO-539) Synergizes with Agents Targeting Chromatin Regulation or Apoptosis and Sensitizes AML with MLL Rearrangement or NPM1 Mutation to Venetoclax

MLL1-rearranged (MLL-r) leukemias are dependent on the interaction of the adaptor protein menin with the oncogenic MLL (KMT2A) fusion protein. We previously demonstrated that NPM1 mutant (NPM1mut) acute myeloid leukemias (AMLs) exhibit a similar dependency on the menin interaction with wildtype MLL, a histone 3 lysine 4 methyltransferase. Pharmacologic inhibition of the menin-MLL interaction reverses MEIS1/HOX-driven leukemic gene expression, induces differentiation, and has anti-leukemic activity in preclinical NPM1mut and MLL-r AML models. Ziftomenib is currently assessed in clinical phase I/II trials and explorative efficacy against relapsed / refractory AML has been demonstrated. Here, we characterized the effects of ziftomenib on MLL-r and NPM1mut AML in detail and systematically screened for synergistic drug combination partners. First, we assessed the single-agent effects of ziftomenib on MLL-r (MOLM13, MV411, OCI-AML2) and NPM1mut (OCI-AML3) AML cell lines. Cellular assays demonstrated profound dose-dependent killing after seven days of treatment with IC50 values<25nM. HL60 and NB4 cells that are both wildtype for NPM1 and MLL served as negative controls and were not affected. Similar to previously characterized menin inhibitors (menin-i), ziftomenib uniformly induced transcriptional downregulation of MEIS1, PBX3, FLT3, and BCL2 in these leukemias, while genes upregulated upon ziftomenib were enriched for myelomonocytic differentiation processes. Ziftomenib-induced differentiation was also evidenced by cytology and upregulation of cell surface CD14 and CD11b. To identify synergistic combinations, we designed a screen assessing single and combined effects of ziftomenib and 37 targeted compounds (selected by confirmed preclinical or clinical activity against AML), each at four different concentrations and in a constant 1:4 ratio. We observed significant synergistic effects (combination index<0.5) of ziftomenib in MLL-r and NPM1mut AML, particularly pronounced for agents targeting chromatin regulation & DNA damage (e.g., LSD1, PRMT5, and PARP) as well as apoptosis & cell cycle (e.g. BCL2, MCL1, and CDK4/6). As we have demonstrated with other menin-i, ziftomenib induced transcriptional downregulation of FLT3 in the FLT3-mutant AML setting and also exhibited synergistic activity with the FLT3 inhibitor gilteritinib. As ziftomenib also uniformly downregulated BCL2, we assessed its combination with venetoclax in more detailed response assays and found synergistic growth inhibition compared to single treatment or vehicle control in all tested MLL-r and NPM1mut AMLs. While OCI-AML3 cells are not sensitive to venetoclax, the combination with ziftomenib was synergistic when using prolonged combined agent exposure (5 days) but not with standard 24-48h venetoclax treatment. shRNA-based knockdown of MEN1 (menin) also sensitized MOLM13 to venetoclax. Next, we found by Annexin V staining that the combination enhanced apoptosis compared to single agent treatment. Also, BCL2 homology (BH) 3 profiling revealed that ziftomenib treated MLL-r and NPM1mut AML cells exhibited a significantly enhanced cytochrome c release in response to BIM, PUMA, BAD, and MS1 peptides. These findings suggest that ziftomenib-induced apoptotic priming may contribute to synergistic effects with venetoclax in these leukemias. Next, we assessed the combination in a human MV411-derived leukemic xenograft model in vivo. We found significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment compared to the single agent or vehicle control animals. Single agent and combination assessment of four primary human NPM1mut AML patient samples in a human stroma cell co-culture model also showed the most pronounced anti-leukemic effects for the ziftomenib-venetoclax combination compared to all other controls. In summary, we demonstrated that ziftomenib has significant activity against MLL-r and NPM1mut AML and synergizes with certain agents targeting chromatin regulation and apoptosis. The combination of ziftomenib with venetoclax is particularly synergistic and already available for clinical trial assessment.

CD25 Targeting Eliminates Regulatory T Cells and CD25+ Blasts in Acute Myeloid Leukemia

Background Acute Myeloid leukemia (AML) is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. CD25 Mab (also referred to as RG6292 and RO7296682) is an afucosylated, IL-2 non-blocking human IgG1 antibody, shown to efficiently deplete immunosuppressive regulatory T cells (Tregs) in humans and solid tumour models (Kolben 2021) whilst allowing binding of IL-2 to effector T cells and the induction of anti-tumour adaptive immune responses (Solomon, Amann et al. 2020). CD25 Mab binds to CD25+ target cells and its crystallisable fragment (Fc) to Fc receptors (FcR) expressed on the surface of effector cells, such as FcgRIIIa on Natural Killer (NK) cells, monocytes and macrophages. It mediates killing of target cells through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). CD25 Mab is currently being investigated in a phase I dose escalation monotherapy study (NCT04158583) or in combination with atezolizumab (NCT04642365). In AML patients, a higher frequency of immunosuppressive bone marrow Tregs was observed as compared to that of healthy donors and their abundance correlated with poorer outcome (Szczepanski, Szajnik et al. 2009, Han, Dong et al. 2018, Dong, Han et al. 2020). In addition, CD25 is also expressed on a subset of AML cells and may be restricted to leukemic stem cells (LSC) or cells with a progenitor phenotype (Angelini, Ottone et al. 2015, Bertolini, Kageyama et al. 2018, Aref, Azmy et al. 2020). Considering the critical role of LSC in the propagation of the disease and the high relapse rate in AML, it would be important to understand if CD25 targeting could deepen responses and help prevent relapse in patients. In this study, we hypothesized that CD25 Mab may have a dual mode of action whereby it depletes suppressive Tregs and has a direct cytotoxic effect on CD25+ AML cells. Methods We conducted an in-depth characterization of bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry (up to 39 cellular markers) and RNA sequencing using computational analysis. Moreover, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab on Tregs and CD25+ AML cells, as compared to isotype control antibody. Results We found that the abundance of Tregs and CD25+ AML cells correlated strongly with that of the blood in 11 patients with time-matched samples, indicating that blood samples could be used for the identification of predictive biomarkers. Moreover, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing internal tandem duplication mutations in the FLT3 gene (FLT3-ITD). Furthermore, all four patients treated with a hypomethylating agent in combination with venetoclax displayed detectable levels of CD25+ AML cells. We adopted a patient-centric approach (n=14 patients) to study AML clusters with CD25 expression, due to the high inter-patient heterogeneity. Interestingly, CD25+ clusters most commonly expressed CD34, an LSC marker indicating a preferential expression in immature AML cells. Finally, ex vivo treatment of primary AML patient samples with CD25 Mab led to the specific killing of CD25+ AML cells and Tregs by allogeneic NK cells, as compared to isotype control treatment. Taken together, these results provide a proof of concept of CD25 Mab's dual mode of action in AML patient samples. Conclusions Using high dimensional flow cytometry and computational analysis, we provide a deep characterization of AML patient samples and highlight the heterogeneity in the pattern of expression of common AML targets currently under clinical investigation. We dissected the expression of CD25 on healthy T cells and malignant cells and demonstrated that the blood offers a window into the bone marrow composition and could be used to ascertain Treg and CD25+ AML cells prevalence. Mode of action studies demonstrated that CD25 Mab depletes suppressive Tregs and has a direct cytotoxic effect on the CD25+ AML cells. CD25 targeting represents an attractive target for the treatment of AML, especially in patients where CD25 is expressed on LSC or immature AML cells. Our study warrants further exploration of CD25 Mab as combinatorial treatment with, for instance, FLT3 inhibitors or venetoclax.

Single-Cell Multi-Omics Reveals Immune Microenvironment Alterations in T-Cell Acute Lymphoblastic Leukemia

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that predominantly occurs in children and young adults. Although intensified multi-agent chemotherapy improved overall survival rates, relapsed/refractory T-ALL remains difficult to treat. In addition, these intensive chemotherapies carry a risk for short- and long-term side effects, especially in children. There is an urgent need for novel therapeutic strategies to improve patient outcome. Targeting the tumor microenvironment has been recognized as a promising strategy in many adult tumors with high neoantigen load, but it is thought to be less effective in pediatric and hematologic malignancies as these carry few genetic mutations. However, comprehensive studies of the immune microenvironment are lacking in many pediatric malignancies, including T-ALL, but are urgently required to identify novel therapeutic targets. The aim of this study is to unravel mechanisms of immune evasion in T-ALL and to establish novel immunotherapeutic targets for the treatment of T-ALL. Methods We performed 5’ single cell transcriptome, surface proteome of ~200 proteins and immune repertoire analyses of ~54,000 cells from 10 primary T-ALL pediatric patient samples at initial diagnosis and 4 normal donors using the 10x Genomics platform. Sequencing data was processed using Cell Ranger (reference genome hg38). Quality control was performed using Scrublet (doublet removal), DropletQC (quality control damaged cells) and Seurat (percentage mtDNA < 10% and genes expressed > 200). RNA counts were normalized with sctransform and surface protein antibody counts were centered log-ratio normalized using Seurat. Non-linear dimensional reduction (UMAP) and differential gene/protein expression was done using Seurat and cell type annotation was done using Azimuth in combination with known marker genes. In addition, we analyzed differential cell type abundance (MiloR), the immune repertoire (Immunarch), transcription factor activity (pyScenic) and ligand-receptor activity (NicheNet). Results Our multi-omics analysis revealed changes in the immune cell type composition in T-ALL associated with an immunosuppressive microenvironment. Specifically, we identified a significant enrichment of non-classical monocytes, atypical memory B-cells and double-negative T-cells (CD3+, CD4-, CD8-) in the T-ALL immune microenvironment compared to the normal immune microenvironment. These CD21-, CD11c+ atypical memory B-cells showed high expression of multiple inhibitory receptors (including PD-1, FCRL3/5 and ILT2), suggesting muted BCR-signaling. Transcription factor analysis showed transcriptional activity of known B-cell regulators SOX5, TBX21 and POU2F2, of which SOX5 is known to be highly expressed during late B-cell differentiation stages including atypical memory B-cells. Double-negative (DN) T-cells, which were oligoclonal, were also overrepresented in the T-ALL microenvironment. Correspondingly, immune repertoire analysis of normal T-cells showed decreased TCR diversity (Chao1 index, p = 0.016, Wilcoxon rank sum test) compared to normal donors. Surprisingly, these DN T-cells displayed features of CD8+ T-cell dysfunction, such as high expression of PD-1, TIM3 and LAG3, as well as features of regulatory T-cells, such as high expression of IL10, ICOS and CTLA4. These cells also demonstrated high transcription factor activity of EOMES, NFATC1 and ETV1, which are all associated with a CD8+ T-cell dysfunctional transcriptional network, as well as FOXP3 transcription factor activity, the master transcription factor in regulatory T-cells. Conclusions Multi-omics profiling of the immune microenvironment in T-ALL revealed the presence of several populations of dysfunctional cells associated with an immunosuppressive microenvironment. Studying ligand-receptor interactions analysis between malignant and immune cells will provide the basis for future (pre)clinical studies leading to the development of novel immunotherapeutic strategies for the treatment of T-ALL.

Identification of Global miRNA Targetomes within Human Hematopoietic Progenitors and Acute Myeloid Leukemia Uncovers Unique Molecular Networks Centered on Mitochondria Function in Leukemic Stem Cells

Previous studies comparing transcriptome and proteome profiles of the cells that constitute both the normal and malignant hematopoietic hierarchies have revealed substantial uncoupling of transcript and protein expression levels. Despite the widely established role of miRNAs in regulating many cellular functions, the precise contribution of miRNA-mediated post-transcriptional regulation linking gene expression and proteome signatures of hematopoietic stem cells (HSC) and leukemic stem cells (LSC) remains largely unknown. Previous efforts to characterize miRNA targets have largely relied on computational prediction algorithms, which suffer from high rates of false-positive and negative target predictions and ultimately preclude discovery of non-canonical miRNA-target interactions. In this study, we perform Argonaute 2 (AGO2) crosslinking and immunoprecipitation (CLIP)-seq to experimentally map endogenous miRNA-target interactions in human CD34+ hematopoietic stem and progenitor cells (HSPC) from cord blood and six primary AML patient samples in an unbiased, transcriptome-wide manner (Manakov et al. 2022). Using this approach, we identify hundreds of miRNAs directly ligated to thousands of unique targets from miRNA-target chimeric sequencing reads. We find limited overlap (~30%) between miRNA targets captured by CLIP-seq and computationally predicted targets, underscoring the importance of functional validation of miRNA targets in specific cell types. Surprisingly, we find remarkable similarity between abundant miRNAs from HSPC and AML samples, including miR-142 and miRNAs encoded by the miR-17-92 cluster. However, the targets and the gene regulatory networks controlled by these highly abundant miRNAs are vastly different between HSPC and AML. In HSPC, we find that targets of abundant miRNAs are enriched in cell cycle regulation, histone modification and megakaryocyte/myeloid differentiation. In contrast, targets of the same miRNAs are enriched in protein localization/translation, neutrophil function, regulation of apoptosis and electron transport chain (ETC) pathways in AML. Collectively, we find that the composition of the abundant miRNAs is similar in normal HSPC and AML tumors; however they control divergent gene regulatory networks. To identify miRNA-target networks that play functional roles in LSC, we performed small RNA sequencing in 23 AML patient samples where each sorted fraction was assessed for LSC content by xenotransplantation. We derived an LSC-specific miRNA signature consisting of 5 miRNAs (LSC miR-5 score) predictive of overall survival and capable of stratifying AML patients independent of mRNA expression and other established indicators of outcome. Comparison of CLIP-seq targets of the LSC-specific miRNAs from HSPC and AML patient blasts revealed the existence of AML-specific gene regulatory networks centred on oxidative phosphorylation, ETC and cell-matrix adhesion. We further interrogated the functional significance of one miRNA from the LSC miR-5 score, miR-let-7c. We find that miR-let-7c is highly expressed in LSC-enriched populations of two AML in vitro models that recapitulate the leukemic hierarchy. Enforced expression of miR-let-7c in OCI-AML22 cells resulted in expansion of the LSC-enriched CD34+CD38- population and reduction in the CD34- terminally differentiated blast population. Comparison of miR-let-7c targets from HSPC and AML patient samples revealed the existence of distinct gene regulatory networks. In HSPC, miR-let-7c targets are enriched in cell cycle and regulation of transcription, whereas in AML, mitochondrial ETC, oxidative phosphorylation, and cell-matrix adhesion were the most enriched pathways. Moreover, analysis of miR-let-7c targets identified a high prevalence of transcripts encoded by the mitochondrial (mt) DNA (MT-ATP6, MT-ATP8, MT-ND1, -2, -4, -5, MT-CO1), pointing towards a largely uncharacterized function of miRNAs in regulating transcripts encoded by mtDNA in AML. Collectively, miRNA sequencing in LSC-enriched AML samples and identification of miRNA targetomes in HSPC and AML patient samples have revealed unexpected mechanisms of post-transcriptional regulation in normal and malignant hematopoietic stem cells.

Inhibition of Menin, BCL-2, and FLT3 Combined with a Hypomethylating Agent Cures NPM1/FLT3-ITD/-TKD Mutant AML in a PDX Model

Menin inhibition has antileukemia activity in both mixed-lineage leukemia-rearranged (MLL-r) and nucleophosmin (NPM1)-mutated (NPM1c) AML by suppressing the menin-dependent HOX/MEIS gene signature pre-clinically and clinically. However, co-mutations are common in patients with MLL-r or NPM1c AML and menin inhibition in combination with other targeted agents has potential to enhance efficacy. FLT3 is frequently mutated in MLL-r and NPM1c AML. We previously reported that menin inhibition by SNDX-50469 (SNDX) synergized with BCL-2 inhibition by venetoclax (VEN) in vitro using primary AML patient samples and in vivo in a patient-derived xenograft (PDX) model of NPM1/FLT3-ITD/FLT3-TKD mutated AML. However, cells surviving the SNDX and VEN combination at the end of treatment had increased p-FLT3 signaling (Carter BZ et al., Blood 2021). We therefore investigated whether FLT3 inhibition with gilteritinib (Gil) could enhance the efficacy of co-targeting menin and BCL-2 using the same PDX model harboring NPM1/FLT3-ITD/-TKD mutations. We treated PDX-bearing NSG mice with vehicle control, SNDX, Gil, SNDX + Gil, VEN + Gil, or SNDX + Gil + VEN. CyTOF analysis of bone marrow (BM) leukemia cells at the end of treatment demonstrated that the SNDX + Gil + VEN combination targeted leukemia stem/progenitor cells more effectively than SNDX, Gil, or SNDX + Gil. Furthermore, SNDX markedly reduced BCL-2, BCL-XL, BCL2-A1, MEIS1, and PBX3 proteins invivo, which were further reduced by the combination of SNDX, Gil, and VEN (Fig. 1). All treatment arms suppressed leukemia burden in peripheral blood, spleen, and BM and significantly extended survival (Fig. 2) compared to vehicle-treated controls (median 62 days). SNDX (128 days) was significantly more effective than Gil (90.5 days; P = 0.0001). SNDX + Gil (119 days) did not further improve survival compared to SNDX alone, possibly because the two agents have overlapping effects on FLT3 signaling: Gil inhibits FLT3 activity, while menin inhibition suppresses FLT3 levels. The survival duration of the Gil + VEN (121 days) and SNDX + Gil groups did not differ. Even with the reduced Gil (25 vs. 35 mg/kg) and VEN (35 vs. 50 mg/kg) doses compared to the single agent or two-drug combination groups, the SNDX + Gil + VEN combination extended survival (>235 days), significantly longer than SNDX, Gil, SNDX + Gil, or VEN + Gil. Survival in the 3-drug combination group was much longer than in the SNDX + VEN group (143 days), that we previously reported (Carter BZ et al., Blood 2021). To determine if hypomethylating agents further improve survival, we also treated mice with the SNDX + Gil + VEN + 5-Azacytidine (5-AZA) combination. The median survival duration had not been reached when the experiment was terminated on day-414. Importantly, 2/6 mice in the triple- and 3/5 mice in the quadruple-drug treatment groups lived close to the life expectancy of normal NSG mice with no detectable leukemia cells in spleens and BMs when the experiment was terminated on day-414, suggesting that these combinations potentially eliminated all leukemia cells resulting in cures. Conclusions: Findings demonstrate that the combined inhibition of menin, BCL-2, and FLT3 has profound activity against AML and AML stem progenitor cells with NPM1/FLT3-ITD/-TKD mutations. The combination reduces the HOX/MEIS signature and the antiapoptotic BCL-2 proteins, resulting in major survival benefit. The quadruple combination of SNDX + Gil + VEN + 5-AZA resulted in cure of >50% of mice carrying triple mutant AML. Results warrant the clinical development of concomitant inhibition of menin, BCL-2, and FLT3 combined with a hypomethylating agent in NPM1/FLT3-mutant AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase I Study of Ruxolitinib in Combination with Abemaciclib for Patients with Primary or Post-Polycythemia Vera/Essential Thrombocythemia Myelofibrosis

Introduction and preclinical rationale: Patients with primary and secondary myelofibrosis (MF) have a reduced life expectancy compared to age-matched controls. The prognosis of individual patients is defined by clinical, cytogenetic, and molecular disease characteristics. Several oral Janus Kinase (JAK)-1/2 inhibitors such as ruxolitinib are approved by the US FDA for the treatment of patients with intermediate or high-risk MF based on improvements in symptom burden and spleen volume reduction compared to physician choice of best available therapy. However, these inhibitors often have a limited duration of response, and survival in patients after ruxolitinib discontinuation is poor (median survival of 13 months). Novel mechanism-based therapies for patients with suboptimal response or progression on ruxolitinib are thus needed. Preclinical studies have demonstrated that JAK2 V617F mutations promote transition from G1 to S-phase of the cell cycle via increased expression of CDC25A. Notably, CDC25A expression has been shown to be upregulated in primary MPN patient samples. Additional cell cycle regulators such as CDK6 and Cyclin D have also been demonstrated to play a role in MPN pathogenesis and to drive myeloproliferation. Preclinical studies testing the combination of the CDK4/6 inhibitor ribociclib and ruxolitinib demonstrated reduction in spleen and liver size when compared to ruxolitinib alone in murine models, suggesting synergism (Rampal et al., Clinical Cancer Research 2021). Methods: Based on these promising preclinical data, we hypothesize that combining ruxolitinib with a CDK4/6 inhibitor could have synergistic effects in patients with primary or secondary MF who have an inadequate response to ruxolitinib monotherapy. In this multicenter, phase I dose-escalation trial we evaluate the safety of combination therapy with ruxolitinib and the CDK4/6 inhibitor abemaciclib. Patients with primary or secondary MF with intermediate-1/2 or high-risk disease by DIPSS who require treatment and had an inadequate response to ruxolitinib are eligible for enrollment. An inadequate response will be defined by (I) palpable splenomegaly ≥5 cm below the left costal margin at study entry AND/OR (II) active MPN symptoms [presence of one symptom score ≥5 or two symptom scores ≥3 using the screening MPN-SAF TSS (Emanuel at al. JCO 2012). (Table 1). This study follows a conventional "3+3” dose-escalation design, in which patients on a stable dose of ruxolitinib (10mg or 15mg BID) with sufficient baseline platelet and absolute neutrophil count will be treated with increasing doses of abemaciclib. The pre-study dose of ruxolitinib will be maintained to determine the maximum tolerated dose of the combination of ruxolitinib and abemaciclib (Figure 1). The primary endpoint of this study is to determine the safety and tolerability, and to identify the recommended phase II dose of abemaciclib in MF patients on a stable, standard dose of ruxolitinib. Secondary endpoints include overall response rate (defined as a composite of complete remission [CR], partial remission [PR], and clinical improvement [CI; defined as anemia, spleen or symptom response]), rates of CR, PR, and CI as well as progression-free survival (PFS), overall survival (OS), and duration of response (DOR) as defined in accordance with the modified 2013 consensus definitions proposed by the International Working Group - Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and the European Leukemia Net (ELN). As part of this clinical trial several correlative studies are planned to identify biomarkers predictive of response to ruxolitinib and abemaciclib as well as mechanisms of resistance (e.g., MAP kinase pathway activation). These include evaluation of changes in variant allele fraction of driver mutations, gene expression profile, cell cycle, clonal architecture, cytokine profile, and bone marrow fibrosis, aiming to inform patient selection as well as other alternative combination therapies for future clinical trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pyrimethamine, a STAT3 Inhibitor, Synergizes with Venetoclax and Shows Efficacy in Hypomethylating Agent Resistant MDS and AML

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) arise after accumulation of multiple genetic and epigenetic alterations in hematopoietic stem cells (HSCs). Alterations in HSCs promote malignant transformation and give rise to leukemic stem cells (LSCs). Current therapies for MDS and AML, including recently FDA approved agents such as venetoclax (Ven), a Bcl-2 inhibitor, have failed to eliminate LSCs, therefore, molecular targeting of aberrant LSCs may be a potential strategy for developing targeted drugs capable of eliminating LSCs. Studies have shown that aberrant HSCs exhibit multiple disrupted cellular pathways, including overexpression of the signal transducer and activator of transcription 3 (STAT3) (Shastri, 2017). Elevated levels of STAT3 are observed in MDS and AML patient samples. Interestingly, an increased level of STAT3 in MDS and AML patients is predictive of a significantly adverse prognosis, thus supporting a critical role of STAT3 signaling in MDS and AML. We have previously shown that an antisense oligonucleotide inhibitor of STAT3, gets rapidly incorporated into HSCs and induces apoptosis and downregulation of STAT3 when compared to health controls (Shastri, JCI 2018). An initial screen to identify compounds that could target STAT3 revealed that pyrimethamine (PYR), an FDA approved anti-parasitic drug, inhibits STAT3 transcriptional activity at concentrations readily achieved in patients (Nelson, 2011). Therefore, we are further evaluating the efficacy of PYR for treatment of MDS and AML. HMA and Ven resistance is a critical issue in MDS/AML and can occur due to the persistence of relapse associated aberrant stem and progenitor cells that overexpress STAT3. Hence, we are evaluating the efficacy of PYR in a hypomethylating agent (HMA) and venetoclax (Ven) resistant model. Our in vitro studies show that PYR can inhibit viability by inducing apoptosis in multiple leukemic cells within its therapeutic index. Western blot analysis reveals that STAT3 protein is overexpressed in a Ven resistant MOLM13 AML line, and in a HMA resistant cell THP1 AML cell line. In HMA and Ven resistant leukemia cell lines, PYR can significantly decrease proliferation when combined with azacytidine (P= 0.0109) (Fig 1A) or venetoclax (P= 0.0271) treatments. Remarkably, PYR and Ven drug combination show a strong synergistic interaction in HMA resistant cell line (Z score = 14.523) and Ven resistant cell line (Z score = 11.065). An in vivo study to evaluate the efficacy of PYR shows that PYR treatment leads to a significant decrease of blasts counts in a cell-line derived xenograft (CDX) leukemic mouse model (Fig 1B) compared to a PBS control. In addition, primary MDS patient samples treated with PYR show increased myeloid differentiation compared to healthy controls. Our results show that MDS/AML cell lines, including HMA and Ven resistant leukemia cell lines, are sensitive to PYR treatment, in a dose dependent manner within the therapeutic index of STAT3 inhibition (5-10 uM). Interestingly, PYR with Ven drug combination shows significant reduction in cell proliferation and likely synergy. Our current findings support PYR synergy with Ven and the combination as a possible therapeutic option for patients with MDS/AML after prior treatment with hypomethylating agents for MDS and AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The sWT1+ - Transcript Confers Cytarabine Resistance Via Upregulation of HOXA3 and GATA2 Axis

The Wilms tumor gene (WT1) encodes for a zinc finger transcription factor which manifests as both tumor suppressive and oncogenic activities. Due to 3 translation start sites and alternative splicing at exon 5 and a lysine, threonine, and serine (KTS) motif, WT1 generates 12 major transcripts. WT1 expression is upregulated in ~90% of leukemia samples and is associated with chemoresistance and disease relapse. However, which WT1 transcripts are associated with chemoresistant has not been defined. We characterized that sWT1+/-, but not other transcripts decreased cell growth, downregulated CD71 expression, and conferred cytarabine resistance. Leukemia blast cells with low CD71 expression are relatively resistant to cytarabine, suggesting that CD71 could be used as a biomarker for cytarabine treatment. Subsequent RNAseq analysis revealed two transcription factors HOXA3 and GATA2 to be uniquely upregulated in sWT1+/-, but not in other transcripts. Consistent with the sWT1 +/- phenotype, HOXA3 or GATA2 overexpression decreased cell growth, reduced CD71 expression, and conferred cytarabine resistance. Real time-PCR analysis showed that overexpression of WT1 and GATA2 upregulates each other and HOXA3 expression; whereas overexpression of HOXA3 upregulates GATA2 expression. Furthermore, relapsed leukemia samples express a relatively higher level of HOXA3 and GATA2 compared to de novo AML samples in the BeatAML cohort. GATA2 and WT1 are also coexpressed in AML samples (TCGA AML and BeatAML). Interestingly, WT1 and GATA2 mutations are mutual exclusive in primary AML and MDS patient samples (TCGA AML, BeatAML, and MDS (MSKCC+ IWG, IPSSM, NEJM)). These data indicates sWT1+-, HOXA3, and GATA2 forms a transcript complex that regulate cytarabine sensitivity. As such, we have identified transcription factors and a biomarker for cytarabine sensitivity in AML.

DIACC2010, Sole-in-Class Selective Inhibitor of Kinesin KIF20A, Has Potent Preclinical Efficacy in Acute Myeloid Leukemia

Context: Despite recent therapeutic advances in Acute Myeloid Leukemia (AML) and the decent proportion of complete remission after induction chemotherapy, relapse rate is still high and long-term survival does not exceed 20%. The therapeutic arsenal of AML could benefit from differentiated strategies. Mitotic kinesins are essential regulators of cancer cell replication and migration. The microtubule associated motor protein KIF20A (also called MKlp2), a member of the kinesin-6 family, plays an essential role during cytokinesis and is also involved in the fission of RAB6-positive vesicles from Golgi membrane, therefore contributing to intracellular vesicular trafficking. KIF20A plays a critical role in the development and progression of many cancers, and its high expression is associated with disease progression and poor survival outcome. More specifically, immature hematopoietic cells, exhibit high KIF20A expression, whereas mature peripheral blood cells do not. Herein we describe the preclinical anti-leukemic efficacy of DIACC2010, sole-in-class selective KIF20A inhibitor. Methods: We used cell lines (U-937, KG-1, CEM, KASUMI, SKM-1, MOLM-14, HL-60, MV-4-11, THP1) provided by ATCC and a Cell line-Derived Xenograft models to demonstrate the anti-leukemic activity od DIAC2010. Briefly, NOD.Cg-Prkdc scid/J (NSG) mice were obtained from Charles River France and maintained under specific pathogen-free conditions. Animals were injected via tail vein with 0.2 106 viable human leukemia cells. Mice were treated with 0.9% NaCl or Diacc2010 (200 mg/kg IV; once a week). Daily monitoring of mice for symptoms of disease determined the time of killing for animals with signs of distress. In addition, primary AML samples were obtained after informed consent. Cells were transplanted to NSG mice, collected after the 3rd transplantation and drug tests were performed ex vivo. All animal experiments were performed in compliance with the laws and in agreement with the French Guidelines for animal handling and approved by animal ethics committees (Agreement no. APAFIS#6743). Golgi imaging was performed using anti-GM130 antibody. Cell cycle, Caspase-Glo 3/7 and Annexin V/PI tests were performed according to the manufacturer's protocols. Results: DIACC2010 demonstrated potent and consistent cytotoxic activity in vitro against a panel of 9 human AML cell lines, with median IC50 of 40 nM (range 10-77 nM). In the same experimental conditions, cytarabine (CYTA) had median IC50 of 207 nM (range 4-1580 nM). DIACC2010 was subsequently evaluated in vivo in xenograft models of AML cell lines. DIACC2010 significantly improved overall survival as compared to CYTA in all models, with dose-dependent efficacy as shown in Figure A ( MOLM14 cells. Next, DIACC2010 cytotoxic activity was evaluated in vitro on 8 primary AML patient samples, IC50 ranged from 57 nM to 728 nM across the tested samples. DIACC2010 antitumoral activity is currently evaluated in Patients Derived Xenograft model. In parallel, no in vitro toxicity of DIACC2010 was observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50 µM). On a mechanistic standpoint, DIACC2010-exposed AML cells displayed characteristic Golgi scattering, observed within 1 hour of treatment and majoring at 6 hours confirming target engagement (Figure B). In parallel, we observed a strong block in cell cycle in G2/M after 6 hours of treatment (45% of cells versus 21% in the control condition). This was associated with a high caspase activation that occurred mainly after 24 hours of treatment. This cascade led to cell death assessed by annexin V/PI between 24 and 48 hours after treatment initiation. Nevertheless, caspase inhibitors ZVAD-fmk and Emricasan partially rescued DIACC2010 cell death, although these inhibitors significantly rescued caspase activation induced by Venetoclax, a BCL-2 inhibitor triggering intrinsic apoptosis commonly used in AML treatment. We ruled out necroptosis and ferroptosis as cell death pathways triggered by DIACC2010 using pharmacological inhibitors. These data strongly suggests that DIACC2010 induces a non-redundant cell death pathway that could be advantageously used to kill resistant AML cells. Conclusion: Altogether, these results confirm the relevance of KIF20A-directed therapeutic approaches and support the development of DIACC2010 for the treatment of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activating a Non-Genetic MYC-Regulating Pathway Sensitizes Acute Myeloid Leukemia to Bromodomain Inhibition

MYC (c-Myc) plays a central role in the oncogenic progression and is commonly overexpressed or amplified in more than 50% of human cancers, including acute myeloid leukemia (AML), which is an aggressive disease with over 70% of patients who cannot survive more than five years. Elevated MYC expression is associated with therapeutic resistance and predicts a poorer outcome in AML patients. While numerous lines of evidence have validated MYC as a therapeutic target of AML and many other cancer types, MYC protein itself has been a challenge for therapeutic targeting. Recently, MYC gene transcription has been linked to the bromodomain and extra-terminal domain (BET) family proteins. BET inhibitors (BETi) inhibit MYC expression and have shown promising antitumor activities in preclinical models and are being evaluated in clinical trials. Despite the therapeutic promise, BETi-associated drug resistance and dose-limiting side effects hinder the clinical applications. Therefore, there is a pressing need to understand the mechanisms controlling drug response and resistance to BET inhibitors. To understand the mechanisms that regulate BETi sensitivity, we leveraged a functional CRISPR screen focusing on about 1,700 human transcription factors. We discovered the aryl hydrocarbon receptor (AHR) as a novel regulator of BETi sensitivity in AML. Our results have further identified that a novel AHR-ELMSAN1 axis is critically involved in regulating MYC and BETi sensitivity. Specifically, we show that constitutive or ligand activation of AHR sensitizes leukemia cells to BETi. AHR signaling represses MYC and synergizes with BETi for maximum inhibition of MYC transcription. Ectopic expression of MYC could rescue the anti-leukemia effects induced by combined AHR activation and BET blockade. Mechanistically, our unbiased bioinformatic analysis unexpectedly identified that AHR functions through activating a non-canonical target, ELMSAN1, a less-studied epigenetic factor known as a member of the MiDAC histone deacetylase complex. Depletion of ELMSAN1 leads to upregulation of MYC and blunts AHR signaling-mediated MYC repression and BETi sensitization. Importantly, we demonstrate that AHR agonist and BETi synergistically inhibit MYC and leukemia cell growth in CD34+ primary AML patient samples in vitro and patient-derived xenografts in vivo. In summary, our data suggest that AHR-ELMSAN1 is a novel pathway for MYC repression, activation of which could sensitize AML to BET-targeting agents. As neither AHR nor ELMSAN1 is found mutated in AML, our research may open a new door to new MYC targeting paradigms by activating novel non-genetic MYC repressors.

Defining Clinical and Molecular Biomarkers for Venetoclax-Based Drug Combinations to Augment AML Therapy

Background: The combination of the BCL2 inhibitor venetoclax with azacytidine (Ven+Aza) is efficacious for elderly patients with acute myeloid leukemia (AML) who are unfit for standard chemotherapy. However, the overall and progression-free survival of these patients after 1 year of therapy is approximately 30-40%, and resistance to Ven+Aza, either primary or adaptive through clonal evolution, limits its durability. Therefore, we evaluated new venetoclax combinations to elucidate mechanisms and biomarkers of response and resistance. Methods: We tested a set of 31 venetoclax (Ven) combinations using an ex vivo assay on primary AML patient specimens (n=433, though not all combinations were screened for all samples). Enhanced efficacy was determined using a combination ratio (CR), defined as the sensitivity of the combination (by AUC) divided by the sensitivity of the most potent single agent AUC. Drug combination synergy was evaluated using the highest single agent (HSA) method. Clinical, genetic, and disease status information were collated, and ex vivo combination sensitivities were compared to matched single-agent data by Friedman test, across groups by Wilcoxon rank sum or Kruskal-Wallis test, and with continuous variables by Spearman rank correlation. For patient specimens with RNAseq data collected (n=279 patients), transcriptomic gene signatures of AML tumor cell state (van Galen, Cell 2019) were correlated with combination sensitivity by Spearman rank. Uni- and multi-variate analyses were applied to the full dataset to identify biomarkers of differential sensitivity to each combination. For one combination, Ven plus the JAK1/2 inhibitor ruxolitinib (Rux), additional genome wide CRISPR screens and follow up in vivo patient xenograft and mechanistic validation experiments using AML cell line models were performed. Results: Using Ven+Aza as a benchmark, we found several venetoclax combinations that exceeded both the ex vivo activity and enhanced efficacy observed with Ven+Aza in primary AML patient samples (Fig. 1A), while showing limited to no effect on stromal cell controls. Evaluation of clinical and genetic data with combination sensitivity revealed many significant transcriptomic, genetic, and immunophenotypic associations. The strong bias in sensitivity of Ven with respect to transcriptomic signatures of primitive tumor cell states compared to more mature, monocytic tumor states was unchanged with Ven+Aza and select other combinations. Importantly, this differential sensitivity was overcome when pairing Ven with several new drug partners such as Rux or the p38 MAPK inhibitor doramapimod (Fig. 1B). Uni- and multi-variate analyses further revealed an array of different biomarkers involving clinical and genetic features that correlated with significant differential sensitivity to each combination. Many of these likewise involved markers of cell differentiation state, such as CD14. To elucidate the mechanisms of enhanced efficacy of the Ven+Rux combination, which is currently under phase 1 clinical investigation, in vivo murine patient xenograft models and genome-wide CRISPR screens in OCI-AML2 cells were performed. Ven+Rux demonstrated superior inhibition of tumor growth in an AML xenograft model compared to either single-agent. The Ven+Rux CRISPR screen identified many genes whose knockdown was enriched in combination-resistant cells, including three (MDM2, MED13L, MEIS2) that were confirmed to confer resistance in subsequent immunoblotting and drug sensitivity validation experiments. In comparison, analysis of a control CRISPR screen using Ven alone revealed numerous gene hits in the JAK/STAT signaling pathway as enhancing the sensitivity of Ven. Conclusion: To expand the utility of venetoclax-inclusive combinations, we prospectively profiled a set of drug combinations and identified several with activity that exceeds Ven+Aza in ex vivo assays. Associations of clinical features with sensitivities to these drug combinations provide insight into their effectiveness which may be used to align the most promising combinations with translation to treatment of discrete subsets of AML patients. Together, these findings support opportunities for expanding the impact of Ven-based drug combinations in AML by leveraging clinical and molecular biomarkers of response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapeutic Targeting of EP300/CBP By Bromodomain Inhibition in Acute Myeloid Leukemia

CCS1477 (also known as inobrodib) is a potent and selective inhibitor of the bromodomains of EP300 and CBP, two homologous histone acetyltransferases with critical roles in cellular growth and differentiation. In pre-clinical analyses we found that CCS1477 induced dose-dependent growth inhibition of a wide range of acute myeloid leukemia (AML) cell lines and primary patient samples of diverse molecular subtypes at low nanomolar concentrations. Growth inhibition was predominantly due to cell cycle arrest and differentiation; there was only modest apoptosis. In vivo, in contrast to control mice which all developed short latency AML, all animals with experimentally initiated MLL-AF9 and MLL-AF10 driven AMLs (in a retroviral transduction and transplantation syngeneic model) treated with 30mg/kg daily PO CCS1477 survived to termination of the experiment at 220 days. In subcutaneous xenograft models, there was dose-dependent growth inhibition of MOLM16 AML cell tumours, with 20mg/kg daily for 21 days completely suppressing tumour growth and no tumour regrowth until 14 days following the end of treatment. Mechanistically, we found that 100nM CCS1477 induced rapid and selective depletion of EP300 in particular from MYB binding sites in THP1 AML cells. ChIPseq demonstrated that in steady state the strongest recruitment of EP300 to chromatin in THP1 AML cells was at sites of MYB and CEBPA transcription factor occupancy, although EP300 was also found genome-wide at sites occupied by other factors including RUNX1 and ETS. Following CCS1477 treatment there was, within six hours, selective loss of EP300 at MYB occupied sites, co-localized reduced chromatin accessibility and histone H3K27 acetylation, and stalling of RNA polymerase at subordinate genes with reduced transcription. This was associated with a dose-dependent redistribution of EP300 to alternate sites and up regulation of differentiation genes. In our ongoing phase I/IIa clinical trial investigating the safety and efficacy of CCS1477 in advanced haematological malignancies (NCT04068597), we have observed therapeutically beneficial granulocytic differentiation responses in patients with high-risk myelodysplasia or relapsed AML. For example, a patient with myelodysplasia with excess blasts 2 (MDS-EB2) evolving to AML who had received three cycles of azacitidine without response was treated with CCS1477 50mg PO (once daily, three days/week). Pre-CCS1477 treatment there was profound neutropenia and multiple admissions for sepsis. After CCS1477 treatment the neutrophil count rapidly normalized and, despite a brief admission for splenic infarction in the first week, the patient remained well for 5 months with no infective episodes. Treatment was stopped due to a rising leukocyte count. Similar responses were seen in other patients. In one of these, single cell RNA sequencing of serial blood and bone marrow samples confirmed appearance of mature myeloid cells on CCS1477 treatment and, within the blast population, up regulation of a multilineage differentiation programme and down regulation of a stem cell programme. Reflecting our findings in cell line models, there was also up regulation of genes induced by MYB knockdown and concordant up and down regulation of genes regulated by CEBPA in THP1 AML cells. Finally, we evaluated the activity of CCS1477 in xenograft models in combination with existing standard of care agents. Azacitidine and CCS1477 in particular demonstrated synergistic growth inhibitory activity in the MOLM16 model, as did venetoclax in MOLM16 and MV(4;11) models. These encouraging pre-clinical and early phase clinical findings set the stage for the clinical trial evaluation of CCS1477 in combination with azacitidine and venetoclax.