Primary AML Research Articles

Anti-Leukemic Effects of CK1α Degrader As Monotherapy and in Combination with Targeted Drugs

CK1⍺ is a member of casein kinase 1 (CK1) family, a multifunctional serine/threonine kinase conserved in eukaryotes from yeast to humans. Substrates of CK1⍺ include various proteins important in cancer and regulate multiple survival pathways such as p53, Wnt, autophagy and NF-B signaling pathways. In particular, CK1- serves as an upstream regulator of p53 pathway through regulation of MDM2 and MDMX and degradation of CK1- may prevent MDM2 and/or MDMX mediated inactivation of p53 function, thus facilitate cell death. We identified an early hit compound, distinct from known immunomodulatory imide drugs (IMiDs) such as lenalidomide or thalidomide. In order to identify compounds having distinct characteristic with previously published IMiDs, we designed multiple compounds with a wide range of glue effect based on the binding mode analysis and binding structure of CRBN and known IMiDs. Among them, PinA1 (early lead compound) selectively degraded CK1-, in the nanomolar range with the highest potency. PinA1 induced expression of p53 which led to apoptosis in AML cell lines with wild-type TP53 (MV4-11, MOLM-13, and MOLM-14), but not in TP53 mutated cells (KASUMI-1, and KG-1). Of note, in TP53 wild-type cells, expression of MDM2 and p21 increased as well, which may limit p53-induced apoptosis in these cells. PinA1 also induced CK1- degradation and p53-dependent apoptosis in primary AML cells in vitro. On the other hand, PinA1 had marginal effects on the viability of human peripheral blood mononuclear cells and CD34+ cells, with minimal activation of the p53 pathway at an efficacious dose range for MV4-11 compared to MDM2 inhibitors. PinA1 enhanced apoptosis in combination with FLT3, BCL-2, or MDM2 inhibitors in AML cell lines (MV4-11, MOLM-14) and primary AML cells in vitro. To test the efficacy of PinA1 in vivo, we injected MOLM-14/ luc/GFP or MV4-11/ luc/Thy1.1 into nonirradiated NGS mice. Five or Six days after cell injection, mice were randomized into 4 groups: vehicle, PinA1, and PinA1 plus AMG232 (MDM2 inhibitor), venetoclax, or gilteritinib. Bioluminescence imaging (BLI) demonstrated significantly less leukemia burden in all treated groups than vehicles; leukemia progression was lowest with combination therapy. Flow cytometry with sacrificed mice revealed the lowest leukemia burden in combination therapy, consistent with those from BLI. Western blotting with sorted leukemic cells from bone marrow and spleen revealed CK1- degradation in vivo. PinA1- and other single drug-treated mice had prolonged survival compared with vehicles, and combination therapy extended survival even further. Patient-derived xenograft models from two FLT3-ITD-mutated AML patients also showed potent anti-leukemic effects of PinA1 as monotherapy and in combination with gilteritinib. In conclusion, our study showed that PinA1 selectively degrades CK1α, enhancing expression of p53, which then induces apoptosis in TP53-wild-type AML cells, but not in TP53-mutated AML cells, thereby producing anti-leukemic effects as monotherapy or in combination with targeted therapies. The minimal effect of PinA1 on normal hematopoietic cells may positively contribute to safety in future clinical trials. This novel targeting approach may improve the current targeted therapies in combination once validated.

Low-Dose IA with Venetoclax Regimen As Induction Therapy for Newly Diagnosed Acute Myeloid Leukemia

Abstract Background: The IA regimen (combination of idarubicin and cytarabine) is used as the most common induction of remission therapy for acute myeloid leukemia, with the complete response (CR) rates of up to 70%. Addition of the BCL-2 inhibitor venetoclax(Ven) to chemotherapy can increase apoptosis priming. In order to further improve the remission rate and prolong survival, our center uses a low-dose IA regimen combined with Venetoclax to treat newly diagnosed primary acute myeloid leukemia under 60 years old. Methods: We retrospectively analyzed 15 patients with newly diagnosed AML, who was under 60 years old. Patients received idarubicin (6mg/m2 IV for 3-days) and cytarabine (60mg/m2 IV for 5-days) with oral venetoclax (400mg daily for 2 weeks) for induction, followed by the same chemo-free regimen for consolidation if patients responded to induction therapy. And then analyze the effects of this regimen on complete remission (CR) rate, minimal residual disease (MRD) negative CR rate, relapse-free survival (RFS), overall survival (OS), and adverse events. Findings: Between January, 2021 and June, 2023, 15 patients were treated with this regimen. The median age was 43 years (IQR, 36-43) and the median follow-up was 4.25 (IQR, 2.73-15.6) months. Among all patients, the ORR was 86.7% (95% CI 62, 96), the CR rate was 80.0% (95% CI 55, 93), and the MRD negative rate was 66.7% (95% CI 42, 85). According to 2022 ELN risk classification by genetics, the ORR in favorable risk patients were 50.0% (1/2), intermediate risk patients were 100% (7/7), adverse risk patients were 83.3%(5/6). Those who had epigenetic modification mutations (NPM1, FLT3-ITD, CEBPA) were more likely to benefit from this regimen. The most common grade 3 and 4 adverse reactions were neutropenia, thrombocytopenia, and anemia. Conclusion: As induction and consolidation therapy whose adverse reactions were tolerable, the low-dose IA with Venetoclax regimen produces deep remission for the patients with newly diagnosed primary AML.

Immunosuppression in Isocitrate Dehydrogenase Mutated Acute Myeloid Leukemia

Introduction: Mutations in the isocitrate dehydrogenase (IDHmut) enzymes 1 and 2 are prevalent in a diverse array of human malignancies, including melanoma, glioma, acute immunoblastic T cell lymphoma (AITL) and acute myeloid leukemia (AML,15-20% of patients). IDHmut enzymes produce R-2-hydroxyglutarate (R-2-HG), an oncometabolite with immunomodulatory capability (Notarangelo et al, Science 2022). Immunotherapies in AML outside of allogeneic stem cell transplantation have proven to be largely ineffective, possibly due to immune evasion mechanisms intrinsic to myeloid malignancies. Herein, we aim to characterize the immune repertoire in IDHmut AML patients at the time of diagnosis, as well as the mechanisms by which R-2-HG may suppress immune cell function. Methods: The immune repertoire in cryopreserved peripheral blood mononuclear cells (PBMCs, n=10) and bone marrow mononuclear cells (BMMCs, n=8) of IDHmut AML patients were characterized using full spectrum flow cytometry to evaluate 30 different immune cell markers. T cell activation (CD25, CD69, CD40L, CD134), exhaustion (PD-1, CTLA-4, Lag-3, Tim-3), differentiation (Foxp3, CXCR3, CCR4, CCR6), and memory (CD45RA, CCR7, CD28, CD95) markers were assessed. IDH wild-type (IDHwt) AML PBMCs (n=4), BMMCs (n=12) and healthy volunteer PBMCs (n=6) and BMMCs (n=3) were used as controls. To investigate the effects of R-2-HG on CD4 + T cell activation, primary human naïve CD4 + T cells (CD4 +, CD45RA +) were isolated from healthy donors and activated (anti-CD3/CD28) in the presence of R-2-HG (20 mM) or PBS control. Activation, proliferation and cytokine production were assessed with flow cytometry, intracellular cytokine staining (ICS) and LEGENDplex at 4, 8, 24 and 72 hrs. The OT-II transgenic mouse model was used to assess antigen-specific T cell activation. T cell metabolism at rest and after activation in the presence of R-2-HG was evaluated using SCENITH (Argüello et al, Cell Metab 2021) and 13C 6-glucose isotope labeling coupled with mass spectrometry (MS) performed by the UCLA Metabolomics Center. Results: Full-spectrum flow cytometry analysis revealed a trend for higher CD4:CD8T cell ratio in AML BMMCs (fold=1.95, p=0.229) as well as increased CD4 + T cell activation and exhaustion markers compared to healthy donor BMMCs. No appreciable differences in CD4 + T cell markers were found between IDHwt AML and IDHmut AML, likely due to AML heterogeneity and small sample size. Nevertheless, our preliminary results suggest CD4 + T cell engagement in AML; thus, we chose to investigate how CD4 + T cells are affected by R-2-HG. Primary human naïve CD4 + T cells activated in the presence of R-2-HG exhibited a defect in early activation demonstrated by decreased CD69 expression (n=7, fold=0.773, p=0.001) and concomitant decreased production of IL-2 measured by ICS (n=7, fold=0.403, p=0.0001) and LEGENDplex (n=3, fold=0.557, p=0.046). Proliferation in the presence of R-2-HG was inhibited in a dose dependent manner (n=6, Division Index fold change=0.768, p=0.006 at 20mM). Antigen-specific OT-II CD4 + T cells showed a decrease in proliferation in response to OVA 323-339 (n=3, Division Index fold change=0.620, p=0.004). Given R-2-HG's structural similarity to α-ketoglutarate (αKG), we hypothesized that disruption in CD4 + T cell activation when cultured with R-2-HG may be due to metabolic dysfunction. Indeed, we found a decrease in global metabolism in CD4 + T cells cultured with R-2-HG (fold = 0.678, p = 0.009). MS revealed decreased levels of succinate and malate downstream and accumulation of αKG upstream of α-ketoglutarate dehydrogenase (KGDH) in the tricyclic acid (TCA) cycle, suggesting a defect in TCA flux (fold succinate / αKG ratio =0.134 unactivated, p = 0.029; 0.231 4h activation, p = 0.012; 0.328 24h activation, p = 0.004; Figure 1). Moreover, MS also showed increased amounts of essential amino acids (Ile, Leu, Met, Phe, Thr, Trp, Val, His; Figure 2), suggesting a compensatory reliance on amino acid import and metabolism. Conclusions: By characterizing primary AML samples at diagnosis, we found trending increases in CD4:CD8 ratio and expression of CD4 + T cell activation and exhaustion markers compared to healthy controls. We also demonstrate R-2-HG suppression of CD4 + T cells, potentially via inhibition of KGDH in the TCA cycle. Our studies show R-2-HG in IDHmut tumors can modulate CD4 + T cell function and contribute to immune escape in human AML.

Single Cell Correlation Analysis: A Novel Method to Analyze Single Cell RNA Sequencing Data Identifies a Self-Renewing Subpopulation of Human Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy. Despite early response to standard chemotherapy, 60-70% of patients relapse with treatment resistant disease and die of relapsed disease. Leukemia stem cells (LSCs), the subpopulation of leukemia cells with self-renewal capacity, can recapitulate leukemia to cause relapse. Ongoing work to define therapeutic targets that can block self-renewal would prevent relapse and disease progression in AML. Prior work has established that LSCs express a distinct gene expression profile. We sought to define the gene expression profile of self-renewal at the single-cell level because a precise understanding of the transcriptional basis for self-renewal could provide more effective therapeutic targets. Our group previously demonstrated that activated NRAS (NRAS G12V) mediates self-renewal in the LSC-enriched subpopulation of AML in the Mll-AF9/NRAS G12V mouse model of AML (Sachs 2014). Single cell RNA-sequencing (scRNA-seq) of the LSC-enriched subpopulation identified three distinct transcriptional profiles. Two genes that encode cell surface markers, Cd36 and Cd69, delineate these distinct profiles. Using in vivo leukemia engraftment assays, we found that only the CD36 -CD69 + LSC subgroup repopulates leukemia while the CD36 +CD69 - LSC subgroup does not, demonstrating that self-renewal capacity is limited to the CD36 -CD69 + subset of the LSC-enriched compartment (Sachs 2020). In contrast, the CD36 +CD69 - LSCs were highly proliferative while the CD36 -CD69 + LSCs were poorly proliferative. These experiments show that self-renewal and rapid proliferation are mutually exclusive features of LSCs, as has previously been shown in normal hematopoietic stem cells (Li 2013). Methods and Results We investigated whether primary human AML stem cells express the murine-derived single-cell self-renewal and proliferation gene expression profiles at the single-cell level. Current computational methods to compare single-cell gene expression profiles use arbitrary similarity scores and do not assess the statistical significance of their findings. Therefore, we developed a new computational method called Single cell Correlation Analysis (SCA). SCA is designed to quantitate the expression of a predefined profile in single-cell RNA sequencing data. The quantification of the expression profiles is assessed on a per-cell basis in an unsupervised manner, meaning that SCA does not rely on any pre-sorting or classification of the query cells. The significance of the quantitative metric (i.e. level of expression) is determined by permutation-based false discovery rate testing. SCA can therefore identify cells that express a profile of interest within a single-cell RNA sequencing dataset and incorporates a measure of statistical significance to that assignment. We used SCA to analyze scRNA-seq data from primary human AML samples. We used data from two samples sequenced by our group and previously published data from 16 samples (van Galen 2019). SCA identified cells that express the murine single-cell self-renewal profile in every sample (0.32-11.5%; FDR < 0.25). We sorted two primary human AML samples to isolate LSCs (CD34 +CD38 -) prior to single-cell capture and sequencing. SCA identified cells that express murine LSC self-renewal profile in both of these samples as well (8.9-11.3%; FDR < 0.25). Furthermore, with these analyses, we found that the single-cell profiles of self-renewal and proliferation were mutually exclusive in human LSCs, as they are in murine LSCs and normal HSCs. We performed gene set enrichment analysis (GSEA) and confirmed that the human cells that express the murine single cell self-renewal profile are enriched in previously reported LSC self-renewal gene expression profiles. Conclusion We present SCA, a novel computational method designed to quantitatively assess single-cell transcriptional datasets and evaluate the statistical significance of its results. Using this method, we demonstrate that primary human AML samples express the single-cell profiles of self-renewal that we previously validated experimentally. SCA-based identification of human self-renewing cells will allow us to interrogate novel transcriptional features of these cells in future studies.

Epigenetic Classification of Acute Myeloid Leukemia Revealed By Genome-Wide Chromatin Profiling

Background Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease and is predominantly defined by genetic abnormalities and morphology. However, many lines of evidence suggest the role of epigenetic alterations in the pathogenesis and the classification of AML, whereas no large-scale comprehensive analyses of both genetic and epigenetic alterations in AML have been reported. Methods We enrolled a total of 1,564 primary AML cases (524 from Japanese and 1,040 from Swedish cohorts), whose diagnostic samples were subjected to ATAC sequencing together with targeted capture sequencing using an in-house gene panel including 331 known AML driver genes and an additional 1,158-1,317 probes for copy number detection. We also performed RNA (N=1,398) and H3K27ac-targeted ChIP sequencing (N=80). Normal bone marrow samples from 25 donors as well as 15 remission samples were analyzed by ATAC-seq as a control. Results ATAC-seq revealed approximately 300,000 reproducible peaks in primary AML. Most peaks were found in gene-distal regions, and explained a large variance across samples. Among all peaks, 72% were not detected in control samples and thus, specific to AML. Analysis of the correlations between ATAC-seq accessibility and gene expression revealed a subset of regulatory elements that regulate nearby gene expression; each peak was linked to a median of 1 gene (0-31), while each gene was associated with a median of 11 peaks (0-110). We next estimated the cellular composition of the bulk AML samples by deconvolution analysis using ATAC-seq data. The predominance of hematopoietic stem cell (HSC), monocyte, and erythroid signatures was associated with French-American-British (FAB) subtypes of minimal differentiation/without maturation subtypes (M0/M1), myelomonocytic/monoblastic/monocytic subtypes (M4/M5), and erythroid subtype (M6), respectively, suggesting bulk ATAC-seq of AML samples reflected the differentiation potential of leukemic cells. The ATAC-based clustering identified 11 distinct epigenetic subgroups, including three well-known genetic classes defined by gene fusions, such as PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11. By contrast, some genetic classes defined by a single mutation, such as NPM1 or CEBPA mutation, were further classified into multiple distinct epigenetic subgroups. For example, the NPM1-mutated subtype was separated into four subgroups based on the varying degrees of ATAC signatures associated with HSC and monocytes. Each subgroup of NPM1-mutated AML showed different clinical characteristics such as white blood cell counts, age, and prognosis, and co-mutation pattern in FLT3, DNMT3A, and IDH1/ IDH2 mutations. Gene ontology analysis revealed that the subgroup of NPM1-mutated AML with high HSC signature showed upregulated TGF-β signaling, while one with monocytic signature showed upregulated TNF-α signaling and interferon-γ response. Next, we evaluated transcription factor (TF) binding sites in a genome-wide manner, using TF motifs and foot printing in upregulated ATAC-peaks in each subgroup and revealed distinct profiles of activated TFs in each epigenetic subgroup of NPM1-mutated AML. In the subgroup characterized by high HSC signature, HSC-related TFs that belong to MECOM, HOX, and RUNX families, were more active. By contrast, other TFs, such as IRF, CEBP, and JUN/FOS families, were more enriched in the subgroup showing high monocyte signature. By developing a prediction model for these subgroups of NPM1-mutated AML based on the gene expression profile, we reproduced these subgroups and validated clinical and molecular features in an external Beat AML cohort. Utilizing ex vivo drug sensitivity dataset in the Beat AML, we identified candidate drugs showing subgroup-specific effectiveness such as panobinostat for NPM1-mutated AML with monocytic differentiation. Finally, ChIP-seq analysis identified a number of super-enhancers in the vicinity of leukemia-related TFs and oncogenes. Conclusion Through comprehensive profiling of chromatin accessibility and gene mutations/expression in a large cohort of AML patients, we demonstrate the epigenetic heterogeneity and identify novel AML subgroups that had unique epigenetic and clinical features. Our findings highlight the role of epigenetic alterations in AML pathogenesis and impact on molecular classification of AML.

Computational Approach Based on Independent Drug Action Identifies Clinically Relevant Combinations for Acute Myeloid Leukemia

Background Acute myeloid leukemia (AML) is clinically and genetically heterogenous due to complex co-mutation landscape. Combination therapy has shown effectiveness in overcoming tumor heterogeneity and acquired resistance across multiple cancers including AML. However, the discovery of novel combinations has traditionally relied on cell line-based measurements that often fail clinically due to inter- and intra-tumor heterogeneity of patients. To address this challenge of finding clinically relevant combination therapy for AML, we applied the independent drug action (IDA) principle (Palmer & Sorger, 2017) to computationally predict drug combinations using ex vivo monotherapy responses measured in primary AML patient specimens (n > 1000). Methods We utilized the ex vivo drug sensitivity testing (DST) of primary AML samples from three studies; 1. FPMTB (Malani et al. 2021), 164 patient responses to 515 single agents; 2. Beat-AML (Tyner et al. 2018), 579 patient responses to 166 single agents; 3. Oregon Health & Science University (OSHU), 596 patient responses to 51 single agents and 170 2-drug combinations (Figure 1A). To summarize the multiparametric dose-response relationship into a single metric, drug sensitivity score (DSS) (Yadav et al. 2014) was calculated from the dose-response cell viability data of the samples using the BREEZE pipeline (Potdar et al. 2020) to derive monotherapy efficacy. We modified IDACombo package (Ling & Huang, 2020) to predict the mean combination DSS of every pairwise combination. IDAComboscore was calculated for each predicted combination to determine if it is more efficacious than its constituent monotherapies. The top hits from predicted combinations were selected by filtering for combinations with mean DSS in the top 10 th percentile, and positive IDAComboscore. These combinations were then ranked in descending order of IDAComboscore. For validation, Spearman correlation was applied to compare the predicted DSS against ex vivo DST-derived DSS of 170 combinations from the OSHU dataset. Results By applying IDA-based prediction on FPMTB and Beat-AML datasets, we identified that the mean combination DSS exceeded monotherapy DSS (p < 2.2x10 -6, Wilcoxon rank-sum test), suggesting that the predicted combination would outperform monotherapy. After accounting for monotherapies that displayed pan-toxicity across healthy samples and primary AML samples (mean DSS > 95 th percentile), we found venetoclax as the top combination partner of the top 100 most effective predicted combinations (FPMTB, n = 33; Beat-AML, n = 35). We further interrogated the makeup of venetoclax-based combinations and found kinase inhibitors (PI3Ki, MEKi, mTORi), and epigenetic modifiers (BETi, HDACi) as common partners. A total of 8 (FPMTB) and 18 (Beat-AML) out of these 100 combinations were made of FDA-approved drugs (both partners). Next, we investigated the robustness of the predictions across 2 datasets, and we found 1767 overlapping combinations with a strong correlation between their predicted mean DSS (Spearman ρ = 0.74, p < 2.2x10 -6). We finally validated the performance of IDA predictions using ex vivo DST data of 170 unique combinations from 596 primary AML samples. We found that the predicted DSS values of the 170 combinations strongly correlated with the actual ex vivo DST-derived combination DSS (Spearman ρ = 0.76, p < 2.2x10 -6, Figure 1B). Using 75 th percentile of DSS distribution as effective combination cutoff, the approach showed an accuracy of 84.7%. We then asked if disease status of patients affected prediction accuracy and found that IDA approach successfully predicted effective combinations for patients at diagnosis as well as at relapse (diagnosis ρ = 0.74 vs relapse ρ = 0.73). Conclusion We have computationally identified and validated clinically actionable drug combinations for AML using ex vivo monotherapy responses from >1000 tumor specimens based on IDA principle. Irrespective of pharmacological interaction, IDA-based combinations are more efficacious than single drugs in AML primary tumors. Our approach enables the major advantage of identifying effective combinations for the entire patient population as an alternative to personalized combinations. Further associations with genetic makeup and transcriptomics signature can be leveraged to identify suitable combinations.

Lessons of Nature in Trisomy-8 MDS and AML: Determinants of Clonal Drive

The clinical consequences of trisomy 8 (+8) are reflected by its categorization as an intermediate risk cytogenetic abnormality in AML while in MDS it has a more favorable impact. Gain of chromosome (chr.) 8 is a frequent event in other hematologic disorders and its prognostic role seems to be dependent on disease context. Some molecular lesions ( RUNX1, ASXL1) are distinctive of +8 AML and important in delineating its clinical phenotype and prognosis. However, the intrinsic molecular drivers contributing to clonal pathogenesis of myeloid neoplasia (MN) as repercussion of gain of chr.8 remain unclear. We stipulated that the study of clinical, genomic, and transcriptomic features of +8 MN will provide important clues to the pathogenesis of +8 and help refine its clinical implications in a specific clinical context. NGS and clinical data from a cohort of 3971 MDS and 3973 AML patients were reviewed, including normal karyotype MDS/AML (NK MDS or AML), isolated +8 (iso +8 MDS/AML), and non-isolated +8 (non-iso +8 MDS/AML). In addition, RNASeq data were available in a subcohort of patients (82: +8; 617 NK MDS). Expression of chr.8 genes was studied to unmask culprit genes in +8 MN. Compared to NK-AML, iso +8 AML patients were older (76% aged >60 years, P= .0005) and had more dysplastic than hyperproliferative phenotype as they presented with lower WBC (median: 9.5 vs 15.2x10 9/L, P= .008) and platelet counts (median: 49 vs 64 x10 9/L, P= .0002). Iso +8 AML had higher percentages of secondary AML (sAML, 17 vs 11%, P= .001) arising from antecedent MDS (14 vs 9%, P= .002) vs NK AML. As expected, survival analysis showed iso +8-AML to have worse median overall survival (mOS) vs NK AML (20 vs 32 months, P= .0001). Yet, when sub-classifying AML based on disease ontogeny (primary vs secondary), a significant mOS difference was only seen in primary AML (21.5 vs 34.5 months, P= .0002; in sAML, 14.7 vs 24.3 months, P= .3). As for molecular associations, a variety of mutations were more frequent in iso +8 AML vs NK AML, including: lineage transcriptional factors ( RUNX1, 33 vs 17%, P< .0001), chromatin modifiers ( ASXL1 37 vs 13%, P< .0001; EZH2 10 vs 3.4%, P< .0001), splicing factors ( SRSF2 30 vs 13%, P< .0001; U2AF1, 12 vs 3%, P< .0001) and cohesin genes ( STAG2, 13 vs 7%, P= .02). In contrast, mutations in FLT3 (33 vs 42%, = .01), DNMT3A (24 vs 35%, P=.002), NPM1 (14 vs 44%, P<.0001) and CEBPA (7 vs 12%, P= .02) were more frequent in NK AML. We then focused on the iso +8 MDS vs NK MDS groups, and interestingly, found that ASXL1 (52 vs 19%, P< .0001), EZH2 (25 vs 4, P< .0001), RUNX1 (29 vs 12%, P= .0009), and STAG2 (27 vs 6%, P< .0001) were more frequently found in the +8 MDS compared to NK-MDS (similar patten to AML), whereas SRSF2 (19 vs 18%, P= .9) and U2AF1 (9 vs 6%, P=.7) were similar in both. The comparison of AML and MDS then showed that iso +8 AML was more commonly associated with mutations in FLT3 (33 vs 3%, P< .0001), IDH1 (17 vs 5%, P= .03), and IDH2 (25 vs 3%, P< .0001), while iso +8-MDS had higher odds of SF3B1 (19 vs 4%, P= .001) ( Fig1A). We then investigated whether specific transcriptomic features may play a role in +8 MN pathogenesis by targeting the transcriptome of 412 chr.8 genes ( Fig1B). After adjusting for zygosity, we identified 186 genes correlating the change in expression levels with clonality. We further interrogated genes possibly eliciting oncogenic potential with log2 fold change (log2FC) ratio of at least 1.25 to reflect an extra chr.8 copy. We found higher mRNA expression levels of 13 putative culprit genes of known oncogenic potential in iso +8 MDS vs NK MDS including the anti-apoptotic protein BAG4 (LogFC 7.1 vs 5.4, P< .0001), possibly conferring growth advantage, toll-like receptor LY96 (LogFC 3.3 vs 2.0, P< .0001),the tyrosine protein kinase LYN (LogFC 8.4 vs 6.7, P< .0001), human oncogene WHSC1L1/NSD3 (LogFC 8.1 vs 6.5, P< .0001)and long non-coding RNA, PVT1 derived from an 8q24 intergenic region (LogFC 4.0 vs 2.2, P< .0001). In summary, iso +8 MN represents a distinct entity defined by older age and dysplastic phenotype, likely due to genotypic enrichment in RUNX1 mutations, while inversely associated with proliferation genes ( e.g., FLT3 and NPM1 in NK). The key culprit oncogenes implicated in +8 MN may be located on the extra chr.8 itself. Indeed, we found transcriptomic changes pertinent to +8 MDS whose role in driving leukemogenesis and interaction with specific myeloid drivers needs to be confirmed by future experimentations on cellular and murine models.

Results of a Phase 1 Trial Testing the Novel Combination Therapy of Venetoclax and Ruxolitinib in Relapsed/Refractory Acute Myeloid Leukemia Patients

Introduction : A functional small molecule screen on a large cohort of primary AML patient samples revealed that the combination of Ruxolitinib (Rux) and Venetoclax (Ven) exhibited ex vivo efficacy and synergy in both newly diagnosed and R/R AML. This observation motivated a Phase I multi-center trial to evaluate Rux+Ven in R/R AML, for which we now report complete toxicity and efficacy data for 30 subjects. Methods: This study was designed to identify a maximum tolerated or recommended Phase II dose (RP2D) and evaluate the overall safety and preliminary efficacy of Rux+Ven therapy. Eligible subjects were ≥18 years old with R/R AML. There was no restriction on the number of previous therapies or prior exposure to Ven or Rux. We included subjects with prior allogeneic transplant if GVHD was controlled and prior MDS (≥75 years old or having poor cardiac or kidney function) who progressed on a hypomethylating agent (HMA) but had no primary AML therapy. In cohorts of 3 to 4, subjects were assigned to receive Rux+Ven at 1 of 6 dose levels according to the “keyboard” Bayesian toxicity probability interval design applied to a target dose-limiting toxicity (DLT) rate of 30%. DLTs were defined as non-disease-related, Grade ≥3 non-hematologic toxicities, with exceptions for nausea, vomiting, infections, febrile neutropenia episodes, and electrolyte abnormalities that resolved within 48 hours. Hematologic DLT was specified as Grade 4 neutropenia after 42 days in the absence of disease. The DLT evaluation period covered the first cycle. Subjects could continue study therapy after two 28-day cycles if they had a response (ie, CR/CRi or MLFS) or were otherwise deriving clinical benefit. Results : This Phase 1 study has completed enrollment (n=30), with all but one patient off-treatment as of June 2023. Baseline patient characteristics are summarized in Table 1. Median age was 69 (range 29-84), 40% of subjects had ≥3 prior therapies, and 43% had prior Ven. At enrollment, 30% had prior MDS, 24% had mutated TP53 (of 25 patients with available data), and 47% had CD56+ blasts. The numbers of patients treated by dose level (DL) were: 3 on DL0 (200 mg qd Ven; 10 mg bid Rux), 4 on DL1 (400 mg qd Ven; 10 mg bid Rux), 3 on DL2 (400 mg qd Ven; 20 mg bid Rux), and 20 on DL3 (400 mg qd Ven; 30 mg bid Rux), the highest dose level of the study. Per prescribing guidelines, the cycle 1 Rux doses of 15 subjects and Ven doses of 20 subjects were reduced because of a concomitant CYP3A inhibitor. The regimen was well-tolerated. As no DLTs were observed, DL3 is the RP2D. The median (interquartile range) duration of therapy was 55 (36-93) days. The most common grade ≥3 hematologic AEs, regardless of attribution, were thrombocytopenia (50%; which was 30% at study enrollment), anemia (40%; 40% at enrollment), neutropenia (37%; 10% at enrollment), febrile neutropenia (33%), and leukopenia (33%; 7% at enrollment). Six subjects had a Grade 5 AE: 4 were unrelated to study drug (2 death NOS, 1 lung infection, 1 respiratory failure) and 2 were potentially related to Ven (1 sepsis, 1 neutropenic pneumonia). Over the first 2 cycles of study therapy, the Clinical Benefit Rate (CBR; defined as the proportion of subjects obtaining at least stable disease) was 63% (95% CI: 44%-80%). Morphologic leukemia-free state (MLFS) was achieved in 20% (95% CI: 8%-39%) and Composite Complete Remission in 10% (95% CI: 2%-27%). Median overall survival (OS) was 3.7 months (95% CI: 2.3-6.5), with 23% (95% CI: 10%-39%) alive 1 year after commencing Rux+Ven. Two participants were exceptional responders: the first achieving MLFS and receiving 16 cycles of study therapy and the second having a complete remission and remaining on-treatment after 31 cycles. Pre-Rux+Ven CD56 expression on blasts correlated with worse response (all 6 ≥MLFS patients were CD56-; Fig 1) and overall survival (HR=2.37, p=0.044; Table 1). Correlative studies such as ex vivo drug assessment, genomic analysis, CyTOF, and single-cell imaging will be presented to enhance this finding. Conclusion: The novel, all-oral combination of Rux+Ven in R/R AML patients was well-tolerated with no DLTs and an encouraging 63% CBR over cycles 1-2. Negative CD56 was a biomarker of response, although additional work is required to determine if this is a broad association with Ven-based therapies or unique to the Rux+Ven combination. The tolerable toxicity profile of Rux+Ven makes it a promising combination to test with HMAs in high-risk AML patients.

ABC Regimen (Azacitidine, Venetoclax and Chidamide) Is Comparable to the “3+7” Regimen in Newly Diagnosed AML: A Real-World Experience from South China

Background: Traditional “3+7” intense chemotherapy (IC) regimen is the standard treatment backbone for fitness patients with newly diagnosed AML. The azacitidine plus venetoclax (AB) regimen has recently been recommended for elderly AML patients or those ineligible for IC treatment. However, neither the “3+7” IC nor “AB” regimen unmet the needs ofadequate safety and effectiveness, especially on aspects of early death and some subtypes of leukemia, such as M5, or with RUNX1, FLT3-ITD, TP53 mutation, or with MCL-1 up-expression. Chidamide, a novel oral histone deacetylase inhibitor, can overcome the upregulating of MCL-1 expression induced by venetoclax, and synergy with azacitidine and venetoclax induces apoptosis of acute myeloid leukemia cells. Azacitidine-Venetoclax -Chidamide (ABC) combination regimen had been successfully used for refractory relapsed AML and newly diagnosed AML-M5 by Prof. Bin Xu and Prof. Shengli Xue, respectively. Whether the ABC regimen can be comparable to the “3+7” IC regimen in newly diagnosed AML induction therapy is still unknown. Aim: This pilot study aims to evaluate the efficacy and safety of the ABC regimen versus the “3+7” IC regimen in newly diagnosed AML. Method: A total of 112 patients with newly diagnosed AML (not APL) were enrolled in this study. Thirty-one patients from the ABC Collaboration Group were unfitness or unwilling to undergo IC therapy and received ABC regimen treatment from May 2022 to July 2023. Eighty-one patients from GDPH in the AML Collaboration Group of South China were treated with a “3+7” (IC) regimen between November 2010 and August 2013. Treatment protocol: The ABC-28d protocol consists of azacitidine 75mg/M 2, d1-7; venetoclax 100mg d1, 200mg d2, 400mg d3-28, and Chidamide 10mg, d1-6/week for 2 weeks, 28 days as one cycle. The ABC-14d regimen consists of azacitidine 75mg/M 2, d1-7; venetoclax 100mg d1, 200mg d2, 400mg d3-14, and Chidamide 5mg, d1-6/week for 2 weeks, 28 days as one cycle. “3+7” IC regimen consists of idarubicin 10mg/M 2, d1-3; cytarabine 100 mg/M 2, d1-7. Baseline characteristics for patients are listed in Table 1. We compared the complete response (CR) and overall response rate (ORR= CR+ CR with incomplete recovery of blood counts CRi + partial response PR) after the first course of treatment, cumulative response rate, and adverse events between these two cohorts. Results: The outcome data of the ABC regimen was updated as of July 2023 for a median follow-up of 78 days (range 28- 402) for the ABC cohort. The ORR and CR/CRi rate of the ABC cohort after the first cycle therapy was not significantly different from the IC cohort (80.6% vs 75.3%, P =0.108 and 71% vs 65.8%, P=0.552). And the cumulative response rate of the ABC cohort was 87.1%. In primary AML, the CR rate of patients in the ABC cohort was higher than the IC cohort (84% vs 65.8%, P=0.075). Of concern, all 12 patients with M4/M5 treated with the ABC regimen achieved CR (100% vs 76.2%, P=0.03). In secondary AML, the ABC regimen was also poor (CR/CRi rate 33.3%). The ABC regimen is not only applicable to the elder patients (CR rate 100%, 15/15) but also young patients(<60 Y) same as the IC regimen ( CR rate: 71.4% vs 65.8%, P=0.726). The response was summarized in Table 1 and Figure 1. Up to 30 July 2023, the ABC cohort has not reached the median remission duration and overall survival (OS). Grade 3 or 4 hematologic adverse events occurred in 81.2% of patients. Neutrophil and platelet median recovery times were 21 days(0-58) and 16 days (0-58), respectively. Patients in the ABC-14d cohort had lower rates of hematologic AEs than ABC-28d cohort (Neutrophil: 12.13±14.33 vs 26.33±16.89, P =0.024 and Platelet: 11.88±13.92 vs 24.58±18.59, P = 0.048). The most frequent non-hematologic AEs of any grade were neutropenic fever (n=15), pneumonia (n=8) and reversible nephrotoxicity (n=4). In the ABC cohort, no patient experienced early death at week 4 or 8. Conclusions: This preliminary study showed that the safety and efficacy of the ABC regimen were comparable to the traditional “3+7” regimen for newly diagnosed AML, even better for the subtype of AML-M4/M5. The ABC-14 days regimen showed comparable efficacy but better tolerability. However, whether the ABC regimen can replace the traditional intense chemotherapy needs to be confirmed by further randomized controlled trials.

Targeting Mitochondrial Calcium Uptake to Eradicate Venetoclax-Resistant Acute Myeloid Leukemia Stem Cells

Introduction Previously, we demonstrated that human leukemia stem cells (LSCs) are preferentially reliant on oxidative phosphorylation (OXPHOS) for survival. Furthermore, inhibition of BCL2 with venetoclax (ven) acts to suppress OXPHOS, leading to the eradication of LSCs. While the activity of ven in targeting LSCs is well-established, nearly all patients eventually develop disease that is resistant to treatment. Thus, in the present study, we sought to better understand the biological properties of ven-resistant LSCs. We reasoned that the expression of known BCL2 interacting proteins in ven-sensitive (ven-S) vs. ven-resistant (ven-R) would provide insights on resistance mechanisms. Initial findings showed that one of the most prevalent molecular features observable in ven resistant LSCs is up-regulation of calcium signaling. As calcium is a critical modulator of multiple enzymes involved in OXPHOS, we hypothesized that the maintenance of high mitochondrial calcium could contribute to circumventing the effects of ven treatment. Thus, the present study was designed to test the functional role of calcium in ven-R LSCs and to determine whether targeting mitochondrial calcium transport represents a therapeutic vulnerability. Methods CITE-Seq analysis was performed on 25 AML patient specimens and analyzed using TotalVI. All studies were conducted in primary human AML specimens with LSCs defined by a ROS-low phenotype. Mitochondrial calcium measurements were done using Rhod2-AM. OXPHOS activity was measured through seahorse and enzyme activity assays. Genetic perturbations were done using siRNA via electroporation and confirmed with western blot. LSC function was assessed by colony formation assays and PDX experiments. PDX experiments were also used to determine efficacy of tumor reduction in ven-R AML samples. Results To identify unique features of ven resistant LSCs, we performed single cell RNA-seq on known ven-S vs. ven-R specimens. As shown in Fig 1, resistant LSCs had higher expression of genes involved in calcium transport into the mitochondria, which was reflected by increased steady-state mitochondrial calcium levels compared to ven-S LSCs. Thus, we investigated targeting of the calcium uniporter, MCU, as a means to reduce mitochondrial calcium levels. Treatment of primary LSCs with pharmacological MCU inhibitors (Ru265, MCUi4), as well as genetic inhibition of MCU with siRNAs, led to decreased mitochondrial calcium levels, suppression of calcium dependent TCA cycle dehydrogenase activity, reduced OXPHOS activity, and impaired LSC function as measured by colony assays (CFU) and engraftment in immune deficient mice. Importantly, MCU inhibition did not significantly impair normal stem/progenitor cells. To develop a clinically relevant system, we investigated bio-available agents known to inhibit MCU. Intriguingly, the only known FDA-approved direct MCU inhibitor is the chemotherapy agent, mitoxantrone (mitox). We performed a series of studies to assess the impact of mitox on ven-R LSCs. Our findings demonstrate that venetoclax resistant LSCs are exquisitely sensitive to mitox. In CFU assays, doses as low 1.0nM show strong inhibitory activity. Further, treatment of immune deficient mice bearing human xenografts (Fig 2), showed significant reduction of bulk disease in primary mice, and strong suppression of LSCs in secondary transplants. Notably, DNA damage was not present in LSCs treated with mitox as measured by gamma H2AX staining. Additionally, parallel studies with the related agents, doxorubicin and etoposide, showed no inhibition of CFU, mitochondrial calcium, or OXPHOS activity. Lastly, no discernable effect on normal hematopoietic stem cell activity was evident. Conclusions Our findings suggest that ven-R LSCs have adapted to their unique cellular demands by increasing basal mitochondrial calcium levels to drive OXPHOS. Based on this finding, we propose that inhibition of mitochondrial calcium uptake impairs multiple enzymes required for OXPHOS, which in turns leads to eradication of LSCs. Importantly, we also show that the well-established chemotherapy agent mitoxantrone can effectively inhibit MCU at nanomolar concentrations and is a highly potent against ven-R LSCs. We are currently working to establish a clinical trial in which low-dose mitox will be deployed to augment ven-based therapy.

Targeting ADSS2 Enhances BH3 Mimetics Treatment Induced Mitochondrial Apoptosis in AML Cells

Recently, the BH3 mimetic-Bcl2 inhibitor venetoclax (VEN), has been approved by FDA in 2018 for the treatment of patients with AML in combination with a hypomethylating agent (HMA) such as azacytidine (AZA). This synergy is likely due to HMA-induced mitochondrial vulnerability associated with cytoplasmic RNA metabolism. Nonetheless, most AML patients treated with the combination eventually relapse, possibly due to persistence of tumor cells not dependent on antiapoptotic proteins (Bcl2, Mcl1). There is a pressing need to develop targeted therapeutics sensitizing AML cells to mitochondrial apoptosis, particularly for those patients failed from VEN associated treatment. To achieve the goal, we performed a CRISPR/Cas9 Knockout (KO) screen (using Bim peptide-based dynamic ΔBH3 profiling as read-out) targeting genes (n=534) involved in cytosolic nucleotides metabolism pathways to increase mitochondrial vulnerability in a BH3 mimetic-refractory AML cell line (HEL). That search identified ADSS2, which catalyzes the first committed/rate-limiting step of AMP biosynthesis from IMP, emerged as the top hit, exhibiting highest ΔBH3 priming. Subsequent BH3 priming assays validated that ADSS2 KO significantly enhanced sensitivity to BH3 mimetics across various AML cell lines, including MOLM13, SKM1, OCI-AML3, and HEL cells. ADSS2 KO also led to a noteworthy reduction in the IC50 values of both VEN and the Mcl1 inhibitor S63845 in these cell lines. Additionally, the synergistic effect between sgRNA targeting ADSS2 and VEN or S63845 was also confirmed in Cas9 expressing cells from two AML patient-derived-xenograft (PDX) samples. We next transplanted AML cells (form one PDX samples) with or without ADSS2-KO into NGS mice, then treated mice with VEN. Notably, VEN treatment (100 mg/kg, QDx14, PO) significantly reduced tumor burden in ADSS2-KO AML mice compared to WT AML mice. Additionally, ADSS2 KO combined with VEN treatment group displayed a survival advantage compared to single VEN treatment group. Further mechanistic investigations revealed that ADSS2 KO led to a remarkable depletion of AMP rather than ATP, and a robust repression of AMPK activity in HEL cells. To assess whether AMPK inhibition mimics the effect of ADSS2 KO, AMPKα-deficient (Mx1-Cre/Prkaa1 fl/fl/ Prkaa2 fl/fl) MLL-AF9 (MA9) transformed murine AML cells were assessed. Indeed, AMPKα depletion substantially enhanced VEN treatment efficiency ex-vivo. Notably, enforced overexpression of a constitutively active AMPKa2 (T172D) construct in a VEN sensitive cell line SKM1, promoted cell resistance to mitochondrial apoptosis by VEN or S63845. Moreover, T172D expression can also abolish ADSS2-KO mediated mitochondrial vulnerability to VEN or S63845 treatment, indicating the pivotal role of AMPK signaling in mediating ADSS2 function. There are no reported ADSS2 inhibitors. To explore ADSS inhibitor, we conducted a fragment-based virtual screen of 8,000,000 compounds (MolPort) library and requested the top-ranking hits for ADSS2 catalysis evaluation by ELISA. Notably, the top two hits AS104 and AS71 exhibited the most potent inhibitory effects (AS104 IC50=2.43µM, AS71 IC50=3.85µM). NMR-STD analysis was conducted in a cell-free system, which unequivocally confirmed the physical binding of inhibitors and the ADSS2 protein. To further investigate the interactions between AS71 and AS104 with ADSS2 in a cellular context, both compounds were subjected to a cellular thermal shift assay (CETSA). Notably, treatment with AS71 and AS104 led to substantial shifts in ADSS2 protein thermal stability, providing evidence of their interactions with ADSS2 within the cellular environment. In both HEL cells and a primary VEN-resistant AML samples, treatment with AS71 and AS104 significantly increased ΔBH3 priming. Collectively, our observation revealed that targeting ADSS2 is critical for sensitizing AML cells to BH3 mimetics. We are now conducting preclinical assessments of ADSS2 inhibitors.

Factors Impacting Response to Olutasidenib in Patients with mIDH1 Acute Myeloid Leukemia

Introduction. Olutasidenib is an FDA-approved, targeted therapy for IDH1-mutated (mIDH1) AML. Among 147 patients with relapsed/refractory mIDH1 AML, 51 (35%) achieved a complete response (CR) or CR with partial hematologic recovery (CRh) after treatment with olutasidenib, with a duration of response of 25.9 months. We conducted sub-analyses to better understand which factors might be predictive of response to therapy. Methods. Patients were divided into groups based on achieving an overall response (CR, CRh, CRi, PR, MLFS) or having best response of stable disease or progression. Groups were compared to determine if there were baseline patient or disease characteristics that might predict attainment of an overall response to olutasidenib. The study was not powered to statistically evaluate subpopulations. Descriptive statistics are provided, and differences of 20% or more between groups were noted. Data cutoff date is June 2021. Results. As of data cutoff, 71 (48%) patients had achieved an overall response; the remaining 76 patients had stable disease (SD), clinical benefit (CB), disease progression (PD) or were not evaluated (due to short time on trial). Composite CR (CR, CRh, CRi) was achieved in 66/71 (93%) of overall responders, with PR and MLFS comprising only 3% of overall responders ( Table 1). With a median follow-up of 10.2 months (range 0.2-38.1), 20 responders were continuing treatment with ongoing responses at the time of data cutoff while 51 had discontinued; reasons for discontinuation were HSCT (n=14 all of whom had ongoing responses), disease progression (n=24), adverse event (n=8), physician decision (n=4) and protocol deviation (1). Responses to olutasidenib therapy were observed across a broad range of patient demographics and disease characteristics, most of which did not appear to impact the rate of response to olutasidenib ( Table 2). Bone marrow blast count at baseline correlated with response, with 63% of patients with baseline counts ≤40% achieving an overall response compared with 34% in those with baseline counts >40%. More patients with R132C (58%) and R132L/G/S (48%) mutations achieved an overall response compared to those with an R132H mutation (26%) However, a previous analysis showed that patients with the R132H mutation were more likely to have a FLT3 and/or NPM1 mutation, which are associated with a poor prognosis (de Botton S, et al, Blood 2021). Although fewer patients in the overall responder groups had FLT3 or NPM1 mutations compared with the non-responder group, 12/38 (32%) with NPM1 and 5/16 (31%) with FLT3 had an overall response. In addition, 2/5 (40%) patients with the TP53 co-mutation had an overall response to olutasidenib. While group outcomes for specific characteristics of disease may suggest a worse prognosis, individual patients treated with olutasidenib may achieve an overall response despite the impact of these negative factors. Two patients in this analysis had mIDH1-R132H mutations as well as FLT3 and NPM1 co-mutations and primary relapsed AML. One achieved a CRh and proceeded to transplant after a DOR of 15.5 months, and the other achieved a CRi lasting 1.3 months and then progressed. The baseline bone blast count was 53% in the first patient and 80% in the second. Two of the responders in this analysis had mIDH1-R132C and a TP53 co-mutation with refractory AML; one achieved a CR and proceeded to transplant after a DOR of 6 months, and the other had a PR and progressed after 3 months. The baseline bone blast count was 25% in the first patient and 65% in the second patient. Conclusions. This post-hoc analysis demonstrated that response to olutasidenib occurred across a wide variety of patient types and disease characteristics, including those generally associated with a poor prognosis. Baseline bone blast count was strongly predictive of response to treatment. Further evaluation of these observations is needed.

Indicators of Response to the Wee1 Inhibitor Adavosertib in Acute Myeloid Leukemia

Wee1 is a nuclear kinase that regulates cell cycle progression by inhibiting Cdk1, which is essential for G2 to M phase transition. Inhibition of Wee1 leads to high Cdk1 activity, causing cells to bypass proper DNA repair at the G2/M checkpoint, resulting in mitotic catastrophe and cell death, plus increased Cdk2 activity, causing abnormal DNA replication and DNA double-stranded breaks. Inhibitors of Wee1 are being investigated for the treatment of a variety of cancers including AML. In this study, we aimed to identify biomarkers for Wee1 inhibitor (Wee1i) response in AML using ex vivo drug sensitivity data, bulk RNA sequence, exome sequence, and associated clinical data for a large set of primary AML samples tested against the Wee1i adavosertib. Mononuclear cells from bone marrow aspirates (n=136) or peripheral blood samples (n=11) were collected from AML patients using approved protocols and after informed consent. 101 samples were collected at the time of diagnosis and 35 samples were from relapsed/refractory (R/R) patients. For ex vivo drug-sensitivity testing, freshly isolated cells were tested against a library of up to 634 oncology drugs (215 approved drugs) including adavosertib. The drugs were pre-plated in 5 concentrations in a 10,000-fold concentration range and incubated with cells for 72h. Cell viability was assessed using the CellTiter Glo (CTG) assay, and a drug sensitivity score (DSS) calculated based on the area under the dose response curve. Somatic alterations of the drug tested samples were identified via exome sequencing of DNA from the AML samples and matched skin biopsy controls. Gene expression data were obtained from bulk RNA sequence data of the AML samples. Out of 136 samples analyzed, the median DSS for adavosertib was 8.45. We found positive and significant correlations between sensitivity to adavosertib and two factors: 1) malignant cell percentage (R= 0.31, p-value= 0.0097), and 2) final relative viability percentage (R=0.58, p-value= 4.3e-13). Correlation analysis between the DSS of adavosertib and 633 other drugs revealed several strong positive and significant correlations. These included drugs with specific mechanisms of actions including HSP90 inhibitors (n=5), JAK2 inhibitors (n=4), topoisomerase II inhibitors (n=4), CDK4/6 inhibitors (n=3), mitotic inhibitors, vinca alkaloid microtubule depolymerizers (n=3), and PLK1 inhibitors (n=3). Since adavosertib also targets PLK1 in addition to Wee1, the correlation with PLK1 inhibitor activity confirms the similar target profiles. Based on the specific adavosertib DSS thresholds, the samples were separated in two groups. Samples sensitive to adavosertib (n=35) had DSS between 21.3 (max) and 11.9 (3rd quartile). Samples resistant to adavosertib (n=34) had DSS between 0.0 (min) and 5.65 (1st quartile). Differential gene expression analysis between the sensitive vs resistant samples showed that multiple isoforms of the PCDH and HOXB genes were upregulated in adavosertib sensitive samples. The GLI2, PI15, and SKIDA1 genes were also upregulated in the sensitive samples (Figure A). In contrast, genes such DNTT, PF4V1, TRH, CD1B, S100A16, and CD34 were overexpressed in resistant samples (Figure B). Gene set enrichment analysis revealed positive enrichment of pathways related to MYC targets v2 in the sensitive samples. On the other hand, hallmark pathways associated with cell cycle, apoptosis, RAS signaling, and p53 showed significant negative enrichment scores in the sensitive samples. No statistically significant associations were observed between drug response and frequently occuring somatic mutations in our dataset. However, genetic abberations related MLLT4-MLL/MLL-fusions were only observed in sensitive samples. Collectively, our current findings show that there may be specific gene expression signatures associated with Wee1i sensitivity such high expression of PCDH and HOXB. Also, the positive enrichment of the MYC target v2 pathway in adavosertib sensitive samples suggests that highly proliferating tumor cells might be more sensitive to Wee1i. In addition, high CD34 expression in resistant samples suggests that immature stem and progenitor cells may be less susceptible to Wee1i. In summary, targeting of Wee1 in AML - as studied in NCT05682170 azenosertib study - may be effective for a molecularly defined subset of patients.

Multimodal Atlas of Paired Diagnosis and Relapse AML Samples Enables Novel Therapeutic Targeting of Surface Antigens

Note: A.H. and M.U. share co-first authorship. AML is an aggressive clonal malignancy characterized by combinations of chromosomal abnormalities, gene mutations, and cell surface antigen (Ag) expression profiles. This heterogeneity contributes to refractory or relapsed disease that presents a major challenge when treating AML. Previous single cell sequencing studies of primary AML samples have provided remarkable insight into the clonal architecture of AML cell populations and how their mutational, transcriptomic, and surface Ag profiles vary between patients. However, information addressing clonal shifts during progression from diagnosis to relapse in large cohorts is lacking. Here, we adapted single cell RNA sequencing with surface Ag feature barcoding to analyze more than 450,000 cells from 28 paired AML patient bone marrow mononuclear cell samples collected at diagnosis and relapse (56 samples). To our knowledge, it is the largest and most comprehensive single cell AML atlas to date. This atlas contains rich clinical metadata including cytogenetics, mutation status of canonical AML genes, treatment history, and survival information. We leveraged these data to identify potential correlations with clonal heterogeneity during progression to relapse and to propose novel strategies for targeting the surface of AML cells. The feature barcoding panel consists of a comprehensive list of 81 surface Ags reported in clinicaltrials.gov or mined from literature, including large proteomics mass spectrometry datasets of AML patient bone marrow samples (deBoer et. al. Cancer Cell 2018; Jayavelu et al, Cancer Cell 2022). Feature barcoding produces antibody derived tag (ADT) counts which are interpreted as relative values but alone does not indicate absolute number of Ags per cell. We addressed this limitation by measuring absolute Ag density of AML Ags, CD33, CLL1, CD123, and EMR2 in patient samples using the flow cytometric QuantiBRITE assay. Focusing on these four Ags, we modeled the relationship between ADT expression and antibodies per cell (ABC) Ag density from QuantiBRITE. The model was applied to impute absolute Ag density for the remaining 77 Ags in all samples. UMAP visualization after sample transcriptome integration revealed distinct clustering of CD45-dim myeloblasts, T cells, B cells, and erythroid cells. Longitudinal analysis identified 28 surface Ags that were differentially expressed (absolute log 2FC > 1, p < 0.01) between relapse and diagnosis myeloblasts across multiple patients (Fig. 1A). An additional 19 Ags were differentially expressed in no more than one patient while the other 19 had no significant change in any patient, highlighting the inter-and intra- heterogeneity of AML at diagnosis and relapse in these patients. Targeting multiple antigens simultaneously may help address the issue of antigen heterogeneity of tumor cells and help avoid potential antigen escape. Because multi-targeting therapies are a potential solution to this, weleveraged the single-cell resolution of our atlas to systematically identify Ags that in combination provide maximum coverage and are significantly co-expressed in myeloblasts. From this analysis, we found 48 and 52 co-expressed Ag pairs in diagnosis and relapse blasts, respectively (r > 0.4, p < 0.01; Fig. 1B). Of note, CD44 and EMR2 were co-expressed at both diagnosis (r = 0.49 p < 0.01) and relapse (r = 0.3, p < 0.01) with >90% of their blasts expressing CD44, EMR2, or both at targetable antigen levels of >1000 Ags/cell. Furthermore, 10 samples had >40% of their blasts co-expressing both Ags simultaneously at >1000 Ags/cell at diagnosis or relapse. 6 of these patients had a >10% increase in co-expression at relapse, 3 had >10% decrease at relapse, and one patient had no co-expression change. Our comprehensive profiling of AML has provided high resolution insight on cell surface at diagnosis and during disease progression and produced novel hypotheses about targetable combinations of surface Ags based on their expression changes between timepoints, co-expression, and surface densities. Furthermore, our reference atlas can be further dissected to characterize the transcriptome and mutational profiles of myeloblasts at diagnosis and relapse thereby prodding an important resource to create surface antigen protein - transcript correlations within AML blasts and to trace molecular signatures during disease progression.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

Loss of Function MBD4 Mutation Impairs DNA Repair in Association with TET2 in Acute Myeloid Leukemia

Background: Methyl-CpG binding domain 4 (MBD4) is a known DNA glycosylase involved in critical steps of base excision repair (BER) and DNA mismatch repair (MMR) and is essential for reducing genotoxic stress in normal cells. MBD4 binds to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) - oxidation products of TET2 dioxygenases - and may initiate a multi-step BER/MMR around mCpG/hmCpG DNA. Loss of function (LOF) mutations in MBD4 (MBD4 MT) result in compromised BER/MMR, leading to genomic instability. Certain germline MBD4 variants have been reported to be predisposing factors for early onset AML, among other cancers. 1,2 In cases screened at our institution, biallelic MBD4 p.R431* & p.L563* tracked with AML in brother and sister. 3 This inspired us to hypothesize that partial or complete loss of MBD4 may result in reduced BER/MMR function and ultimately in genomic instability and faster accumulation of somatic lesions favoring malignant evolution. Methods: We performed NGS and epigenetic studies coupled with detailed biochemical analyses in CRISPR engineered cell lines and primary AML cells and in vivo model systems to dissect the mechanism of clonal evolution of MBD4 MT hematopoietic stem and progenitor cells (HSPCs). Results: We analyzed WES data from 1,692 patients with myeloid neoplasms (MN, 732 MDS; 960 AML). After adequate filtering based on frequency in healthy population (odds ratio of >1.5), 16 variants in MBD4 were identified in 24 MN cases (MDS=9, AML=15). Among them, 4 heterozygous variants of unknown significance of suspected germline nature (p.R425Q, p.N461S, p.R486K, p.D504H) mapped in the glycosylase domain. Comparing MBD4 MT vs. wild-type ( MBD4 WT) cases, MBD4 MT were found in younger patients (median 55 vs. 71 yrs; p = 0.012) with TET2 MT co-occurring at a lower frequency (8 vs. 24%). Since MBD4 is known to preferentially bind with 5hmCpG DNA, a stable epigenetic mark induced by TET2, we investigated the biochemical basis of MBD4 dysfunction and its connection to LOF TET2 MT. We show that MBD4 hydrolase activity levels affect the genomic accumulation of 5hmC in a TET2-dependent manner. MBD4 KO led to more than 2-fold increased 5hmC in TET2WT cells but not in TET2KO cells, suggesting a functional connection between MBD4 and TET2 in BER/MMR. To test the function and effects of the 4 variants (p.R425Q, p.N461S, p.R486K, p.D504H) identified in our cohort on the activity and function of MBD4, we generated these mutations in a pcDNA plasmid using site directed mutagenesis. The mutants were ectopically expressed in isogenic CRISPR-engineered MBD4 KO HEK293 cells and its effects on 5hmC accumulation were tested. The impact on the functional outcomes were compared to WT controls as described earlier. 4 We found that these 4 variants lead to MBD4 protein instability, and thereby lower MBD4 DNA glycosylase activity resulting in increased (1.6 to 3.3-fold) genomic accumulation of TET2-dependent oxidation products. To establish the effect of MBD4 MT in primary cells, we generated CRISPR-engineered human CD34 + HSPCs and assessed their clonal evolution via colony forming assay. Loss of MBD4 led to clonal advantage reflected in persistent colonies in MBD4KO CD34 + HSPCs that demonstrated myeloid differentiation bias similar to what was observed in patients, suggesting that MBD4 MT may favor clonal evolution to fully blown MN. Finally, transcriptomics analysis of MBD4KO in K562 cells in the presence and absence of functional TET2 demonstrated loss of BER genes compared to WT. The co-immunoprecipitation of MBD4 followed by western blot analysis identified XRCC1, a key component of the BER complex, as a part of MBD4 interactome. Comprehensive characterization of protein complexes with LCMS/MS in K562 cells suggests that MBD4 is involved in maintaining genomic integrity. Consistent with our hypothesis, MBD4 KO in K562 and THP1 cells resulted in a 2- and 3-fold increase in spontaneous mutations respectively, as observed via HPRT assay. Conclusions: Genomic instability due to MMR/BER dysfunction in MBD4 MT cells may result in a higher rate of clonal progression. Interestingly, MBD4 deficiency may provide opportunity for therapeutic targeting of DNA repair pathways in a subset of MBD4 MT-associated MN. Indeed, our findings indicate the possibility of leveraging MBD4 as an actionable target to regulate DNA methylation in association with TET2 during leukemogenesis.

FT538, iPSC-Derived NK Cells Are Potent Inducers of Apoptosis in AML Cells and Their Effect Is Synergistic in Combination with Approved Therapeutic Strategies

Background: Natural killer (NK) cell-based therapies have been increasingly studied with their advantages of less treatment-related toxicity and lack of HLA restriction. Induced pluripotent stem cell (iPSC)-derived NK cells offer an opportunity for a standardized, off-the-shelf treatment with the potential to treat a wider patient population. FT538 cells are triple gene-edited iPSC-derived NKs that express a high affinity non-cleavable CD16 maintaining constant activity, a CD38 knock out preventing CD38 antibody-induced fratricide, and an IL-15/IL-15R fusion preventing the need for cytokine administration, the main source of side effects in NK cell-based therapies. Several studies have shown success in treating AML with NK cell therapy, but the effect of iPSC-derived NK cell therapy in AML is unexplored. We hypothesized that FT538 cells may be able to target high-risk AML that is traditionally resistant to most chemotherapeutic agents and synergize with Venetoclax, 5-azacytidine, Cytarabine, and Gilteritinib. Methods: To examine the effect of the FT538 cells on AML cells, we co-cultured FT538 cells at ratios of 2:1, 4:1, and 8:1 with 8 AML cell lines: OCI-AML2, OCI-AML3, THP1, U937, Molm-13, Molm-14, MV4-11, and Kasumi-1, and with cells from 11 different AML patients. We analyzed the NK cell-induced apoptosis through an Annexin-V binding assay using IncuCyte live cell imaging. As Molm-13 and MV4-11 cell lines are FLT3 mutated, to determine the combination effect of FLT3 inhibitors with FT538 cells, we treated Molm-13 and MV4-11 cells with Gilteritinib for 24 hours before seeding the FT538 cells and co-culturing overnight. The effects of the combination were then analyzed through flow cytometry with a Beckman Coulter CytoFlex. Finally, to determine the combinatorial effect of the FT538 cells with approved therapies, we treated OCI-AML3 and U937 cell lines with Venetoclax, Azacitidine, and Cytarabine. After treatment, we performed flow cytometry to measure the leukemic cell death following the combinatorial therapies. Results: We observed FT538 NK-mediated killing in all the leukemic cell lines tested. A two-way ANOVA determined that an increase in the FT538 to AML cell ratio resulted in an increase in NK cell-mediated killing of AML cells (p<0.0001). Interestingly, the number of apoptotic AML cells was 1.5 to 6.1-fold higher in the OCI-AML2, OCI-AML3, U937, and THP1 cell lines than in the Molm-13, Molm-14, MV4-11, and Kasumi-1 cell lines (p<0.0001). This could be related to the FLT3 mutations in the Molm-13, Molm-14, and MV4-11 cell lines or to the TP53 mutation in the Kasumi-1 cell line. Further, to validate this finding in primary AML cells, we observed a ratio-dependent effect on FT538-mediated killing of AML cells. However, we did not observe any statistical difference by mutation, though this could be related to the limited sample size for specific mutations. Finally, flow cytometric analysis of the effects of combination treatment demonstrated that the FT538 cells synergize with Venetoclax, or Cytarabine, or Azacitidine, or Gilteritinib (p<0.05) in the killing of AML cells. This could be because NKG2DL upregulation has been shown in AML cells treated with Venetoclax and FLT3 inhibitors and in Cytarabine-resistant AML cells, and the NKG2D/NKG2DL pathway is a main mechanism of NK cell-mediated killing. Conclusion: iPSC-derived NK therapy offers a standardized, off-the-shelf option for NK cell therapies. FT538 iPSC-derived NK cells induce apoptosis in AML cell lines and patient samples in a dose-dependent manner and show a synergistic effect with Venetoclax, Gilteritinib, Azacitidine, and Cytarabine. Therefore, iPSC-derived NK cell therapy may be a promising possibility for AML treatment, particularly for patients resistant to standard therapies.

MicroRNA Expression Patterns Reveal a Role of the TGF-β Family Signaling in AML Chemo-Resistance.

Resistance to chemotherapy is ultimately responsible for the majority of AML-related deaths, making the identification of resistance pathways a high priority. Transcriptomics approaches can be used to identify genes regulated at the level of transcription or mRNA stability but miss microRNA-mediated changes in translation, which are known to play a role in chemo-resistance. To address this, we compared miRNA profiles in paired chemo-sensitive and chemo-resistant subclones of HL60 cells and used a bioinformatics approach to predict affected pathways. From a total of 38 KEGG pathways implicated, TGF-β/activin family signaling was selected for further study. Chemo-resistant HL60 cells showed an increased TGF-β response but were not rendered chemo-sensitive by specific inhibitors. Differential pathway expression in primary AML samples was then investigated at the RNA level using publically available gene expression data in the TGCA database and by longitudinal analysis of pre- and post-resistance samples available from a limited number of patients. This confirmed differential expression and activity of the TGF-β family signaling pathway upon relapse and revealed that the expression of TGF-β and activin signaling genes at diagnosis was associated with overall survival. Our focus on a matched pair of cytarabine sensitive and resistant sublines to identify miRNAs that are associated specifically with resistance, coupled with the use of pathway analysis to rank predicted targets, has thus identified the activin/TGF-β signaling cascade as a potential target for overcoming resistance in AML.

Abnormal adipogenic signaling in the bone marrow mesenchymal stem cells contributes to supportive microenvironment for leukemia development

BackgroundAcute myeloid leukemia (AML) is an aggressive hematological malignancy, associated with unfavorable patient outcome, primarily due to disease relapse. Mesenchymal stem cells (MSCs) residing in the bone marrow (BM) niche are the source of mesenchyma-derived subpopulations, including adipocytes, and osteocytes, that are critical for normal hematopoiesis. This study aimed to characterize BM-derived adipocyte/osteocyte fractions and their crosstalk with AML cells as a potential mechanism underlying leukemogenesis.MethodsBM cell subpopulations derived from primary AML patients were evaluated using humanized ex-vivo and in-vivo models, established for this study. The models comprised AML blasts, normal hematopoietic stem and progenitor cells and mesenchymal stromal subpopulations. ELISA, FACS analysis, colony forming unit assay, whole exome sequencing and real-time qPCR were employed to assess the differentiation capacity, genetic status, gene expression and function of these cell fractions. To explore communication pathways between AML cells and BM subpopulations, levels of signaling mediators, including cytokines and chemokines, were measured using the ProcartaPlex multiplex immunoassay.ResultsThe study revealed deficiencies in adipogenic/osteogenic differentiation of BM-MSCs derived from AML patients, with adipocytes directly promoting survival and clonogenicity of AML cells in-vitro. In whole exome sequencing of BM-MSC/stromal cells, the AHNAK2 gene, associated with the stimulation of adipocyte differentiation, was found to be mutated and significantly under-expressed, implying its abnormal function in AML. The evaluation of communication pathways between AML cells and BM subpopulations demonstrated pronounced alterations in the crosstalk between these cell fractions. This was reflected by significantly elevated levels of signaling mediators cytokines/chemokines, in AML-induced adipocytes/osteocytes compared to non-induced MSCs, indicating abnormal hematopoiesis. Furthermore, in-vivo experiments using a fully humanized 3D scaffold model, showed that AML-induced adipocytes were the dominant component of the tumor microenvironment, providing preferential support to leukemia cell survival and proliferation.ConclusionsThis study has disclosed direct contribution of impaired functional, genetic and molecular properties of AML patient-derived adipocytes to effective protection of AML blasts from apoptosis and to stimulation of their growth in vitro and in vivo, which overall leads to disease propagation and relapse. The detected AHNAK2 gene mutations in AML-MSCs point to their involvement in the mechanism underlying abnormal adipogenesis.AaFEVfq6u-1YqH7bnQ85uaVideo

2289P Determining anti-cancer efficacy of a reversible LSD1 inhibitor, EXS74539, in primary AML tissues with limited thrombocytopenic effects

2289P Determining anti-cancer efficacy of a reversible LSD1 inhibitor, EXS74539, in primary AML tissues with limited thrombocytopenic effects