Acute Myeloid Leukemia Samples Research Articles

AML/T cell interactomics uncover correlates of patient outcomes and the key role of ICAM1 in T cell killing of AML.

T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human-engineered cytotoxic CD4+ T cells. Single-cell RNA-seq of primary AML samples and CD4+ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4+ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro. Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1, a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing by primary ex vivo-isolated CD8+ T cells in vitro, and engineered CD4+ T cells in vitro and in vivo. While AML heterogeneity implies that multiple factors may determine their sensitivity to T cell killing, these data show that ICAM1 acts as an immune trigger, allowing T cell killing, and could play a role in AML patient survival in vivo.

CD37 is a safe chimeric antigen receptor target to treat acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells in the bone marrow and the peripheral blood. Nearly half of the AML patients relapse after standard induction therapy, and new forms of therapy are urgently needed. Chimeric antigen receptor (CAR) T therapy has so far not been successful in AML due to lack of efficacy and safety. Indeed, the most attractive antigen targets are stem cell markers such as CD33 or CD123. We demonstrate that CD37, a mature B cell marker, is expressed in AML samples, and its presence correlates with the European LeukemiaNet (ELN) 2017 risk stratification. We repurpose the anti-lymphoma CD37CAR for the treatment of AML and show that CD37CAR Tcells specifically kill AML cells, secrete proinflammatory cytokines, and control cancer progression invivo. Importantly, CD37CAR Tcells display no toxicity toward hematopoietic stem cells. Thus, CD37 is a promising and safe CAR Tcell AML target.

Targeting CD33+ acute myeloid leukemia with GLK-33, a lintuzumab-auristatin conjugate with a wide therapeutic window.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of AML blasts, making it an attractive target for therapy of acute myeloid leukemia (AML). While previous CD33-targeting antibody-drug conjugates (ADCs) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novel ADC with improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linker-payloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated anti-tumor activity at single dose as low as 300 µg/kg in mice, while maintaining tolerability at single dose of 20 - 30 mg/kg in rats. In contrast to both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Transcriptome free energy can serve as a dynamic patient-specific biomarker in acute myeloid leukemia

Acute myeloid leukemia (AML) is prevalent in both adult and pediatric patients. Despite advances in patient categorization, the heterogeneity of AML remains a challenge. Recent studies have explored the use of gene expression data to enhance AML diagnosis and prognosis, however, alternative approaches rooted in physics and chemistry may provide another level of insight into AML transformation. Utilizing publicly available databases, we analyze 884 human and mouse blood and bone marrow samples. We employ a personalized medicine strategy, combining state-transition theory and surprisal analysis, to assess the RNA transcriptome of individual patients. The transcriptome is transformed into physical parameters that represent each sample’s steady state and the free energy change (FEC) from that steady state, which is the state with the lowest free energy.We found the transcriptome steady state was invariant across normal and AML samples. FEC, representing active molecular processes, varied significantly between samples and was used to create patient-specific barcodes to characterize the biology of the disease. We discovered that AML samples that were in a transition state had the highest FEC. This disease state may be characterized as the most unstable and hence the most therapeutically targetable since a change in free energy is a thermodynamic requirement for disease progression. We also found that distinct sets of ongoing processes may be at the root of otherwise similar clinical phenotypes, implying that our integrated analysis of transcriptome profiles may facilitate a personalized medicine approach to cure AML and restore a steady state in each patient.

Differential prognostic values of the three AKT isoforms in acute myeloid leukemia

The PI3K-AKT-mTOR pathway lies at the confluence of signaling pathways in which various components are subjected to activating genetic alterations in acute myeloid leukemia (AML), thus contributing to oncogenesis. Three AKT isoforms exist in humans. However, whether one isoform predominates in AML remains unknown. This study reveals that AKT3 behaves very distinctly than AKT1 or AKT2 in both normal myeloid differentiation and AML. During normal differentiation, AKT3 is preferentially expressed in hematopoietic stem cells whilst AKT1 becomes preferentially expressed as cells differentiate into granulocytes or monocytes. AKT2 expression remains unchanged. In AML, AKT3 expression varies widely among patient samples and is counterintuitively high in mature/monocytic leukemia. Furthermore, a low level of AKT3 expression is strongly correlated to genetic alterations associated with a better outcome (NPM1 mutations and RUNX1-RUNX1T1 translocation), while a high level is correlated to alterations associated to a bad outcome (RUNX1 mutations; and SRSF2, U2AF1, SF3B1, ASXL1 and BCOR mutations occurring frequently in MDS and MPN). Consistently, a high AKT3 expression level appears as a very strong predictor of poor survival. Curiously, although modestly varying among AML samples, a high AKT1 expression shows in contrast as a strong predictor of a better patient outcome. These data suggest that AKT3 and AKT1 expressions have strong, yet opposite, prognostic values.

Identification of Nonfunctional Alternatively Spliced Isoforms of STING in Human Acute Myeloid Leukemia.

We find that AML acquires resistance to innate immune activation via the STING pathway through aberrant splicing of the STING transcript including two novel forms described herein that act as dominant negatives. These data broaden understanding of how cancers evolve STING resistance, and suggest that the AML tumor microenvironment, not the cancer cell, should be the target of therapeutic interventions to activate STING.

Abstract 3352: A CRISPR-enabled function genomics profiling platform in acute leukemia: Updates from a pilot study of Function (Fx) Heme utilizing fresh patient samples

Abstract Introduction: In this study we utilized a novel primary cell CRISPR/Cas9 profiling approach to identify gene dependencies in fresh acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patient samples. The primary aim of this prospective study was to demonstrate the feasibility of the CRISPR-based personalized functional genomic testing with fresh patient samples in the clinical setting. Methods: AML and ALL patient peripheral blood samples (10-16 mL) were obtained at time of initial diagnosis and/or at relapse, and if feasible post-treatment. Primary cells were transduced with lentivirus harboring Cas9 enzyme and a ‘drug targets’ sgRNA panel. Transduced cells were harvested at early (input) and Day 8 timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Results: 37 patients were included in the study, 23 with AML, 9 with Philadelphia (Ph) (+) ALL, 5 with Ph (-) ALL. The majority of patients had adverse or poor risk disease. The average age of patients was 52.6, range 18-84. 23 were male, 14 female; 20 identified as white, 17 as other. 20 patients were sampled at initial diagnosis, 12 were sampled at time of relapse and 5 at time of refractory disease. Serial samples were collected from 9 patients. 34 patients received treatment following sample collection. The majority of patients received intensive chemotherapy or a hypomethylating agent with venetoclax, however 15 patients received some form of targeted therapy. At the time of the abstract submission, Fx Heme results were available for 23 patients and among these, we identified potential treatment targets in 10 samples (43%). Importantly, 60% of the positive samples have a dependency on a gene clinically targetable by an approved leukemia drug. In addition, our profiling platform enabled the discovery of novel druggable dependencies in 100% of the positive samples. Conclusions: With this pilot study we demonstrated that utilizing small volume fresh patient samples to assess CRISPR-defined gene dependencies on targeted therapies in AML and ALL is feasible, timely, and results in clear Fx Heme dependency gene output. Our data indicates that CRISPR-based comprehensive functional profiling has the potential to add valuable information beyond traditional NGS methods to determine the optimal therapy for patients with AML and ALL. Future directions include translating further into the clinical setting and correlating Fx Heme dependence with response to targeted treatment in acute leukemia patients. Citation Format: Kaitlyn C. Dykes, Tuyet R. Tan, Karl Hodel, Kathleen Tran, Maci Meyers, Leonardo Hagmann, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath, Tiffany N. Tanaka, Stephane Corbel. A CRISPR-enabled function genomics profiling platform in acute leukemia: Updates from a pilot study of Function (Fx) Heme utilizing fresh patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3352.

Abstract 7011: The DNA methylation landscape across the TCR loci genes in patients with acute myeloid leukemia

Abstract The T-cell receptor (TCR) repertoire is partly shaped by epigenetic modifications, including DNA methylation. Hypomethylating agents like decitabine are used to treat patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The precise DNA methylation patterns within the TCR loci and their dynamics associated with HMAs treatments remain underexplored. Here, we analyzed methylation patterns across TCR alpha and beta gene loci in T cells from GSE175758 and GSE67170 datasets, comparing patterns between patients with AML and healthy controls. We also investigated the impact of decitabine on the methylation patterns of TCR gene loci in patients with AML and MDS (GSE175758 and GSE80762).The overall levels of methylation between T cells from healthy donors and patients with AML were significantly different within the TRAV (0.55 vs 0.42, P &amp;lt; 0.001) and TRBV regions (0.70 vs 0.59, P &amp;lt; 0.001) with 46 TRAV and 33 TRBV sites differentially methylated positions, respectively. However, methylation patterns in HSCs (GSE63409), T cells from healthy donors and patients with AML and those in AML cells were strongly correlated (R &amp;gt; 0.67, range 0.67-0.99, P &amp;lt; 0.001).Because decitabine preferentially acts on leukemic cells and given the high correlation levels between AML cells and T cells across datasets we analyzed the effects of decitabine on TCR DNA methylation patterns in AML cells. In the leukemic cells decitabine treatment led to significant demethylation across 33 and 30 CpG sites within the TRAV and TRBV genes, respectively. In the pre-treatment AML samples higher methylation beta values were observed in differentially methylated positions (DMPs) compared with non-DMPs (TRAV: 0.847 vs 0.407; P &amp;lt; 0.001; TRBV: 0.771 vs 0.538; P = 0.049). In samples largely enriched of myeloid leukemic blasts, we found that TRAV and TRBV loci methylation patterns differ significantly between AML patients with good, moderate, and poor risk patients (P &amp;lt; 0.001). We found TRBV6-1 position 1 and 2 methylation was lower in patients with good risk, compared with intermediate risk (pos1: 0.07 vs 0.13, P=0.04; pos2: 0.1 vs 0.2, P = 0.002) and poor risk (pos 1: 0.07 vs 0.21, P &amp;lt; 0.001; pos 2: 0.1 vs 0.32, P &amp;lt; 0.001) and was lower in patients with intermediate risk compared with poor risk (pos1: 0.13 vs 0.21, p=0.007; pos 2: 0.21 vs 0.32, P = 0.001). The high correlation of TCR DNA methylation patterns between the T cells and the myeloid cells, suggests the need for similar analyses in the context of AML T cells. The study highlights the presence of a conserved TCR loci methylated signatures which is highly correlated between myeloid leukemic cells and T cells and is associated with clinical outcomes, offering a potential avenue for therapeutic intervention. Hypomethylating agents, such as decitabine, can modulate TCR loci methylation patterns. Citation Format: Mateusz Pospiech, John Beckford, Advaith M. Kumar, Mukund Tamizharasan, Jaqueline Brito, Gangning Liang, Serghei Mangul, Houda Alachkar. The DNA methylation landscape across the TCR loci genes in patients with acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7011.

Abstract 7123: Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML)

Abstract Isocitrate dehydrogenase 1 and 2 (IDH1/2) convert isocitrate to α-ketoglutarate in the tricarboxylic acid (TCA) cycle. A missense mutation in IDH1/2 leads to accelerated conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent dioxygenases including Ten-Eleven Translocation (TET) enzymes. TET enzymes are DNA demethylases that convert 5-methylcytosine to 5-carboxycytosine and regulate CpG island promoter methylation signatures. In IDH1/2 mutant glioma, inhibition of TET2 enzyme by 2-HG can lead to promoter hypermethylation of O6-methylguanine methyltransferase (MGMT) and reduce its expression. Approximately 10-15% of de novo or treatment-related AML harbor mutually exclusive mutations in IDH1/2 or TET2, and emerging resistance towards clinical IDH1/2 inhibitors, therefore warrants novel therapeutic options. We performed a pan-cancer TCGA analysis and observed MGMT promoter hypermethylation and reduced expression in IDH1/2 AML. Our independent study on 10 AML patients (5 IDH1/2 and 5 TET2 mutant) corroborated the TCGA data and showed a significantly reduced expression of MGMT in comparison to 7 wild-type AML samples. As MGMT resolves O6-alkylguanine lesions during DNA damage, we performed an exhaustive alkylator screen, where temozolomide (TMZ) and our novel compound KL-50 emerged as the top hits that imparted a maximum therapeutic index in multiple MGMT (MGMT+/–) isogenic AML cell line models. Both TMZ and KL-50 induced MGMT-dependent DNA strand breaks and replication stress and activated ATM and ATR DNA damage response pathways in a time-dependent manner. Inhibition of ATR activation with Ceralasertib, an ATR inhibitor (ATRi) currently in clinical trials for chronic myelomonocytic leukemia and myelodysplastic syndromes, imparted a profound MGMT-dependent synergy with TMZ and KL-50. Researchers have established in glioma that mismatch repair (MMR) loss confers resistance to TMZ, and reported studies also show evidence of MMR loss in refractory or relapsed AML patients. Thus, we developed numerous MMR-deficient isogenic AML cell lines in an MGMT+/– background and observed MMR loss-mediated resistance to TMZ and abrogation of its synergy with ATRi. Unlike TMZ, KL-50 retained a profound synergy with ATRi in an MGMT-dependent manner irrespective of MMR status. We are currently investigating our novel KL-50 and ATRi combination in MGMT/MMR isogenic cell line-based in vivo AML models and in AML patient-derived xenografts to establish it as a promising novel biomarker-targeted therapy for AML patients. Citation Format: Prateek Bhardwaj, Ranjini Sundaram, Amro Baassiri, Martin Matthews, Jennifer VanOudenhove, Susan Gueble, Stephanie Halene, Ranjit Bindra. Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7123.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 618.

Abstract 5178: Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients

Abstract INTRODUCTION: Volasertib, a polo-like kinase 1 (PLK1) inhibitor, has a roughly 30% CR/CRi response rate in de novo acute myeloid leukemia (AML) patients when combined with low-dose cytarabine. However, its development was halted due in part to toxicity in a Phase 3 study. We have built a flow cytometry-based, high-throughput predictive precision medicine platform to forecast clinical responses to various treatments. Here, we present our design plan for a Phase 2a clinical trial with volasertib in relapsed/refractory (R/R) AML using a modified protocol to reduce toxicity and the development of a companion diagnostic to predict responders in a follow-up Phase 2b study. METHODS: We will initiate an open-label clinical trial in AML with volasertib in venetoclax-hypomethylating agent (HMA) R/R patients while co-developing a companion diagnostic to selectively enroll predicted responders in a follow-up, Phase 2b, study. Primary and secondary objectives will focus on efficacy/safety of intravenous volasertib and ex vivo responses, respectively. To reduce the toxicity seen in the Phase 3 study, specific strategies like tailored dosing will be implemented. To develop the clinical trial assay, peripheral blood or bone marrow samples were collected from AML patients and treated ex vivo with volasertib for 3 days. Surviving blast cells were enumerated by flow cytometry. Using a dynamic separation model restricted to the 35% most sensitive samples, we estimated optimal blast cutoffs for volasertib sensitivity. RESULTS: In single-agent experiments with 41 primary AML samples (31 de novo, 9 R/R, 1 with disease status unclear), we observed dose-response profiles consistent with volasertib’s role as a cell cycle inhibitor. Irrespective of the disease status, median and mean EC50 values were about 10 nM (1st-3rd quartile from 7-12 nM). To identify an optimal concentration of volasertib for patient stratification, we compared the ex vivo resistant blast fractions at each concentration with the areas under the dose-response curves (AUCs), assuming that in our case, AUCs most accurately reflect clinical responses. At 31.6 and 100 nM, the Pearson correlation coefficients were highest (r &amp;gt; 0.8), suggesting that these are optimal stratifying doses. With these concentrations and corresponding blast cutoff values, 32-33% of the de novo and 25% of the R/R patients would have been predicted to be responders were they given volasertib, assuming a positive predictive value of 100%. CONCLUSION: Volasertib’s EC50 values clustered within a narrow range, suggesting that EC50 may not be a suitable metric to stratify patients into responders and nonresponders, in contrast to AUCs and residual blast fractions at 31.6 and 100 nM of volasertib, which displayed more heterogeneous distributions. Limitations include small sample sizes, especially for the R/R group. Citation Format: Markus Daniel Lacher, Glenn Michelson, Allen Ling, Wade Anderson, Jeaneille Vinas, Christine J. Gu, Chris Leonardi, Joseph Wagner, Gabriel N. Mannis. Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5178.

Abstract 3904: Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy with a complex clonal architecture that requires in-depth immunophenotyping for disease characterization. Conventional AML ex vivo models for preclinical drug screening lack functional readouts that provide single cell level data to identify drug activity in defined AML subsets with unique immune signatures. Here, we present a novel platform that links Multiparameter Flow cytometry with high-throughput EX vivo drug screening (MFLEX) in AML patient-derived cell models to simultaneously detect phenotypic changes and predict cell-subset specific drug responses in AML. Method: We developed a 21-color high-dimensional immune profiling panel that captures AML heterogeneity by delineating primitive and mature AML blasts along with normal hematopoietic lineages. The panel also includes markers for functional measurement of apoptosis, proliferation, differentiation, and expression of BCL2 family proteins in lymphoid and myeloid cell subsets. Ex vivo culture conditions were optimized using physiologically relevant stroma-free cytokine-rich media that supports primary AML cells with minimal phenotypic shift, allowing for high throughput functional drug sensitivity testing. Timepoints and dosing conditions for AML standard of care (SoC) agents were optimized to support combination screens. Multi-parameter datasets were analysed for functional differences and visualized using dimension reduction methods. Results: We tested MFLEX on clinically annotated AML samples to understand sensitivity profiles to venetoclax and combination partners: 5-azacytidine, cytarabine, fludarabine and gilteritinib. We identified distinct drug sensitivity profiles for venetoclax in AML patients with varying leukemic differentiation states where M0/M1 subtypes were more sensitive to therapy than M4/M5. We also observed cell lineage specific sensitivities, with myeloid blast being more sensitive to venetoclax compared to autologous CD4+ and CD8+ T-cells and this observed difference was correlated with higher expression of BCL2. Our platform demonstrated that patients who previously failed venetoclax therapy could benefit from a combination with 5-azacytidine or fludarabine and this was patient-specific. We further evaluated MFLEX’s predictive value for patient clinical response by systematically comparing venetoclax sensitivity in patient-derived xenograft models in vivo vs ex vivo and found a high correlation between drug responses. Conclusion: We have established a unique, immune drug screening platform, MFLEX, that has high translational value and can be implemented in clinical trials to identify drug response patterns, novel combinations, and potential biomarkers of response for patient stratification in AML. Citation Format: Reecha Shah, Heba Wander, Michelle Hsueh, Muskan Floren, Wei Liu, Patricia Cheung, Courtney Andersen, Michael White, Nathan Kingston, Pablo Morentin Gutierrez, Lisa Drew, Giulia Fabbri, Elena Bibikova, Daniel Auclair. Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3904.

Abstract 3400: Single-cell proteogenomics analysis of AML samples treated with decitabine and venetoclax reveals divergent treatment escape mechanisms

Abstract The combination therapy of hypomethylating agents and venetoclax (HMA+Ven) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist including relapse and treatment resistance, to which our understanding remains incomplete. Previous studies have revealed genetic (RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms. In this context, we hypothesized that integrative assessment of both genetic and phenotypic factors could further unveil the contributors of HMA+Ven resistance. To determine the predictors of treatment response, we first analyzed the baseline characteristics of 208 newly diagnosed, secondary, and relapse/refractory AML patients treated with Decitabine and Venetoclax (Dec+Ven). While complex cytogenetics, KRAS and TP53 mutations were significantly associated with treatment resistance, phenotypic factors such as bone marrow cell compositions or French-American-British (FAB) classifications were not. To identify the factors of resistance at the cellular level, we performed a single-cell targeted DNA-antibody sequencing (DAb-seq) on a subset of 33 patients. In total, 60 multi-timepoint bone marrow samples were analyzed, which included five patients with paired baseline-relapse timepoints. From all five patients, we identified that stem-like cells were largely eradicated and replaced by more differentiated cells such as monocyte-like or erythroid-like cells. At relapse, we identified three patterns of phenotypic shifts: monocytic shift, erythroid shift, and mixed monocytic/erythroid shift. The phenotypic shifts were not always associated with the expansion of new genetic mutations, except for in two cases: one patient had monocytic shift accompanied by the expansion of KRAS mutation, and another patient had erythroid shift accompanied by the expansion of NRAS mutation. To examine the mechanisms behind the phenotypic shifts, we performed a single-cell RNA sequencing on three of the relapsed patients. As a result, we confirmed the upregulation of BCL2L1 in the patient with erythroid shift (N=1), and MCL1 in the patients with monocytic shifts (N=2), but not BCL2 in both. These findings are consistent with known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. To summarize, we conclude both genetic and phenotypic factors play a role in Dec+Ven resistance. Most importantly, we report that divergent phenotypic transitions, from stem-like toward more differentiated phenotypes (monocytic or erythroid), were frequently observed in AML relapse following Dec+Ven therapy. Close monitoring of phenotypic alterations during HMA+Ven treatment may facilitate the precise identification of AML patients predisposed to relapse. Citation Format: Yi June Kim, Zongrui Li, Abhishek Maiti, Tomoyuki Tanaka, Hidetaka Uryu, Ken Furudate, Kala Hayes, Naval Daver, Naveen Pemmaraju, Tapan M. Kadia, Farhad Ravandi, Nicholas J. Short, Maro Ohanian, Gautam Borthakur, Nitin Jain, Ghayas C. Issa, Lucia Masarova, Steven M. Kornblau, William G. Wierda, Sanam Loghavi, Sa A. Wang, Guillermo Garcia-Manero, Hagop Kantarjian, Courtney D. DiNardo, Marina Y. Konopleva, Koichi Takahashi. Single-cell proteogenomics analysis of AML samples treated with decitabine and venetoclax reveals divergent treatment escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3400.

Abstract 862: Multi-omic analysis of allelic expression patterns of imprinted and non-imprinted genes in cancer and their association with drug response

Abstract Malignant cells undergo a broad array of epigenetic changes, including changes in imprinting patterns. In normal cells, imprinted genes have parent of origin-specific monoallelic expression, which is controlled via epigenetic mechanisms. Imprinted gene loci are often complex and frequently include multiple isoforms, which have different individual patterns of parent-of-origin monoallelic or biallelic expression in the normal tissues. Many imprinted genes promote cell proliferation and body growth, and their expression is important during embryonic and postnatal development. Dysregulation of imprinting in tumors has been suggested to contribute to growth and proliferation of cancer cells. Previously we reported that chemotherapy responses of cancer cell lines from a variety of tumors and of patient-derived acute myeloid leukemia samples were associated with copy number, expression, or DNA methylation of selected imprinted genes. In the current project, we examined allele-specific patterns of expression (monoallelic vs. biallelic) of imprinted genes and isoforms in tumors and studied their potential effect on response of cancer cells to drug treatment. We analyzed expression patterns at the whole gene, isoform, and exon levels of 59,283 autosomal protein-coding genes and ncRNA genes, with a special focus on 94 imprinted genes and their isoforms. We developed a computational pipeline to evaluate monoallelic and biallelic expression patterns of each gene in cancer cell lines and in tumor samples based on available matched RNA-seq expression data, whole exome sequencing information, and copy number data. Our initial analysis included 108 cell lines from the Cancer Cell Line Encyclopedia (CCLE), which had been derived from 9 pediatric and adult tumor histologies. We observed variation of allelic expression patterns of imprinted genes and their isoforms in tumor cell lines, including differences across and within cancer histologies. We also observed predominantly monoallelic expression of multiple additional genes. Some of them (PRIM2, BCLAF1, MAP2K3, and SEC22B) had been previously reported as potentially imprinted. Monoallelic expression of other genes identified in our analysis (e.g., GRK2, CBX4, and additional genes) was likely caused by molecular mechanisms other than imprinting. Allelic expression patterns and overall expression levels of isoforms of multiple imprinted and non-imprinted monoallelically expressed genes (e.g., CPA4, MAP2K3, and other genes) were associated with in vitro drug response. We also report our ongoing validation of allelic expression patterns of imprinted and non-imprinted genes, isoforms, and exons in primary bladder and breast tumors and in matched solid normal tissue samples using patient data from the Cancer Genome Atlas (TCGA). These results provide new knowledge about epigenetic dysregulation in cancer. Citation Format: Julia Krushkal, Travis L. Jensen, George Wright, Yingdong Zhao. Multi-omic analysis of allelic expression patterns of imprinted and non-imprinted genes in cancer and their association with drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 862.

Prediction of metabolites associated with somatic mutations in cancers by using genome-scale metabolic models and mutation data

BackgroundOncometabolites, often generated as a result of a gene mutation, show pro-oncogenic function when abnormally accumulated in cancer cells. Identification of such mutation-associated metabolites will facilitate developing treatment strategies for cancers, but is challenging due to the large number of metabolites in a cell and the presence of multiple genes associated with cancer development.ResultsHere we report the development of a computational workflow that predicts metabolite-gene-pathway sets. Metabolite-gene-pathway sets present metabolites and metabolic pathways significantly associated with specific somatic mutations in cancers. The computational workflow uses both cancer patient-specific genome-scale metabolic models (GEMs) and mutation data to generate metabolite-gene-pathway sets. A GEM is a computational model that predicts reaction fluxes at a genome scale and can be constructed in a cell-specific manner by using omics data. The computational workflow is first validated by comparing the resulting metabolite-gene pairs with multi-omics data (i.e., mutation data, RNA-seq data, and metabolome data) from acute myeloid leukemia and renal cell carcinoma samples collected in this study. The computational workflow is further validated by evaluating the metabolite-gene-pathway sets predicted for 18 cancer types, by using RNA-seq data publicly available, in comparison with the reported studies. Therapeutic potential of the resulting metabolite-gene-pathway sets is also discussed.ConclusionsValidation of the metabolite-gene-pathway set-predicting computational workflow indicates that a decent number of metabolites and metabolic pathways appear to be significantly associated with specific somatic mutations. The computational workflow and the resulting metabolite-gene-pathway sets will help identify novel oncometabolites and also suggest cancer treatment strategies.

Cellular abundance-based prognostic model associated with deregulated gene expression of leukemic stem cells in acute myeloid leukemia.

Background: Previous studies have reported that genes highly expressed in leukemic stem cells (LSC) may dictate the survival probability of patients and expression-based cellular deconvolution may be informative in forecasting prognosis. However, whether the prognosis of acute myeloid leukemia (AML) can be predicted using gene expression and deconvoluted cellular abundances is debatable. Methods: Nine different cell-type abundances of a training set composed of the AML samples of 422 patients, were used to build a model for predicting prognosis by least absolute shrinkage and selection operator Cox regression. This model was validated in two different validation sets, TCGA-LAML and Beat AML (n = 179 and 451, respectively). Results: We introduce a new prognosis predicting model for AML called the LSC activity (LSCA) score, which incorporates the abundance of 5cell types, granulocyte-monocyte progenitors, common myeloid progenitors, CD45RA + cells, megakaryocyte-erythrocyte progenitors, and multipotent progenitors. Overall survival probabilities between the high and low LSCA score groups were significantly different in TCGA-LAML and Beat AML cohorts (log-rank p-value = and , respectively). Also, multivariate Cox regression analysis on these two validation sets shows that LSCA score is independent prognostic factor when considering age, sex, and cytogenetic risk (hazard ratio, HR = 2.17; 95% CI 1.40-3.34; p < 0.001 and HR = 1.20; 95% CI 1.02-1.43; p < 0.03, respectively). The performance of the LSCA score was comparable to other prognostic models, LSC17, APS, and CTC scores, as indicated by the area under the curve. Gene set variation analysis with six LSC-related functional gene sets indicated that high and low LSCA scores are associated with upregulated and downregulated genes in LSCs. Conclusion: We have developed a new prognosis prediction scoring system for AML patients, the LSCA score, which uses deconvoluted cell-type abundance only.

The multi-CDK inhibitor dinaciclib reverses bromo- and extra-terminal domain (BET) inhibitor resistance in acute myeloid leukemia via inhibition of Wnt/β-catenin signaling

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions, regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development, but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML, both as monotherapy and in combination. To date, many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9, which has a known physical and functional interaction with the BET protein BRD4. Therefore, we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here, we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines, primary AML samples, and in vivo. Further, we demonstrate novel activity of dinaciclib through inhibition of the canonical/β-catenin dependent Wnt signaling pathway, a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels, including downregulation of β-catenin, the Wnt co-receptor LRP6, as well as many Wnt pathway components and targets. Moreover, dinaciclib sensitivity remains unaffected in a setting of BET resistance, demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately, our results demonstrate rationale for combination CDKi and BETi in AML. In addition, our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML.

CD155/PVR determines acute myeloid leukemia targeting by Delta One T cells

AbstractRelapsed or refractory acute myeloid leukemia (AML) remains a major therapeutic challenge. We have recently developed a Vδ1+ γδ T cell–based product for adoptive immunotherapy, named Delta One T (DOT) cells, and demonstrated their cytolytic capacity to eliminate AML cell lines and primary blasts in vitro and in vivo. However, the molecular mechanisms responsible for the broad DOT-cell recognition of AML cells remain poorly understood. Here, we dissected the role of natural killer (NK) cell receptor ligands in AML cell recognition by DOT cells. Screening of multiple AML cell lines highlighted a strong upregulation of the DNAM-1 ligands, CD155/pulmonary vascular resistance (PVR), CD112/nectin-2, as well as the NKp30 ligand, B7-H6, in contrast with NKG2D ligands. CRISPR-mediated ablation revealed key nonredundant and synergistic contributions of PVR and B7-H6 but not nectin-2 to DOT-cell targeting of AML cells. We further demonstrate that PVR and B7-H6 are critical for the formation of robust immunological synapses between AML and DOT cells. Importantly, PVR but not B7-H6 expression in primary AML samples predicted their elimination by DOT cells. These data provide new mechanistic insight into tumor targeting by DOT cells and suggest that assessing PVR expression levels may be highly relevant to DOT cell–based clinical trials.

Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.