is a member of Scientific Advisory Boards for Kodikaz Therapeutic Solutions, Coherus Biosciences, and Zenshine Pharmaceuticals, and is an inventor on multiple patients related to CD47 cancer immunotherapy licensed to Gilead.
Key Points MEK inhibition rescues T cells from activation-induced cell death in an AML model. MEK inhibitor sensitivity is associated with inflammation pathways and PD-L1 expression.
BCL2 is an antiapoptotic protein commonly expressed in hematologic malignancies. Overexpression of BCL-2 is a poor prognostic factor in acute myeloid leukemia (AML). Venetoclax (ABT-199) is a highly selective BCL2 inhibitor that can induce cell death in multiple leukemia cell lines. Recently, venetoclax received an FDA breakthrough therapy designation for use in combination with hypomethylating agents in treatment-naïve patients with AML who are unfit for intensive chemotherapy. However, venetoclax was only modestly effective as monotherapy in relapsed/refractory AML (19% CR/CRi). The aim of the current study is to integrate genomic and functional screen data to identify biomarkers to predict venetoclax sensitivity and resistance in AML, and to identify potential venetoclax combination treatment strategies. In this study, we investigated approximately 200 AML patient samples and correlated clinical parameters, whole exome sequence data, and RNAseq gene expression data with in vitro drug screening data (drug area under the curve (AUC)) to identify subsets of AML samples with sensitivity or resistance to venetoclax alone and in combinations with 10 small molecular inhibitors (Array-382, dasatinib, JQ-1, idelalisib, quizartinib, palbociclib, panobinostat, ruxolitinib, sorafenib, and trametinib). For gene expression, we observed that venetoclax correlated with 3 gene expression clusters (coefficient frequency: 0.94, 0.80 and 0.71 respectively) among 21 gene expression clusters in AML, associated with innate immune system, neutrophil degranulation, and interleukin-10 signaling. Among the BCL2 gene family, venetoclax AUC positively correlated with BCL2A1 (r=0.59, p<0.0001) and MCL1 (r=0.26, p=0.001) expression, whereas it negatively correlated with BCL2 (r=-0.53, p<0.0001) expression. BCL2A1 is the only BCL2 family gene within all three clusters and correlated the best with venetoclax sensitivity. Interestingly, within the three gene clusters, we observed that cell surface markers CLEC7A (CD369) and CD14 correlated with venetoclax sensitivity (r=0.68 and 0.64, p<0.0001). AML patient samples expressing CD14 detected by flow cytometry also demonstrated reduced venetoclax sensitivity (p=0.005), which could potentially serve as a biomarker to identify venetoclax resistant patients. For cytogenetic categories and mutations, we observed that AML samples harboring PML-RARA translocations, WT1, and FLT3 with IDH1 mutations are more sensitive to venetoclax, and samples with TET2, KRAS, PTPN11 and SF3B1 mutations are more resistant. We validated the effect of WT1, KRAS, and PTPN11 mutations on venetoclax sensitivity by performing drug assays on mouse bone marrow stem cells and/or AML cell lines overexpressing each mutant. Samples harboring PTPN11 mutations demonstrated high MCL1 expression, and PTPN11 mutant-transduced cells remain sensitive to Idasanutlin, which was previously shown to downregulate MCL1 expression. Samples with KRAS mutations demonstrated high BCL2A1 expression, which potentially mediate venetoclax resistance. For venetoclax drug combinations, we observed that venetoclax-trametinib demonstrated a synergistic effect on samples that are sensitive to venetoclax, whereas venetoclax-palbociclib, venetoclax-Array-382, venetoclax-sorafenib, venetoclax-ruxolitinib, venetoclax-dasatinib, and venetoclax-idelalisib are active against samples that are resistant to venetoclax, indicating potential therapeutic combinations. Interestingly, the CDK inhibitor palbociclib demonstrated no effect on the majority of AML samples and does not correlate with the BCL2A1 expression as a single agent, yet shows the most robust synergy with venetoclax, especially on samples that are resistant to venetoclax and with high BCL2A1 expression, indicating a potential synthetic lethal interaction. Venetoclax-palbociclib AUC also negatively correlated with CLEC7A and CD14 expression, indicating that venetoclax-palbociclib could circumvent venetoclax resistance to treat patients with high CLEC7A and/or CD14 expression. In summary, we have identified that CD14 and/or CLEC7A could be used as biomarkers to predict venetoclax sensitivity in AML, and we propose to combine venetoclax and palbociclib to treat patients with a venetoclax resistant profile (high CD14/CLEC7A expression or high BCL2A1 expression, or presence of KRAS mutations). Disclosures Druker: Beta Cat: Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Bristol-Meyers Squibb: Research Funding; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Novartis Pharmaceuticals: Research Funding; Millipore: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Patient True Talk: Consultancy; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Celgene: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Majeti:BioMarin: Consultancy; Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Incyte: Research Funding; AstraZeneca: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Leap Oncology: Equity Ownership; Seattle Genetics: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Syros: Research Funding; Aptose: Research Funding; Agios: Research Funding.
Many acute myeloid leukemia (AML) patients exhibit hallmarks of immune exhaustion, such as increased myeloid-derived suppressor cells, suppressive regulatory T cells and dysfunctional T cells. Similarly, we have identified the same immune-related features, including exhausted CD8+ T cells (TEx) in a mouse model of AML. Here we show that inhibitors that target bromodomain and extra-terminal domain (BET) proteins affect tumor-intrinsic factors but also rescue T cell exhaustion and ICB resistance. Ex vivo treatment of cells from AML mice and AML patients with BET inhibitors (BETi) reversed CD8+ T cell exhaustion by restoring proliferative capacity and expansion of the more functional precursor-exhausted T cells. This reversal was enhanced by combined BETi and anti-PD1 treatment. BETi synergized with anti-PD1 in vivo, resulting in the reduction of circulating leukemia cells, enrichment of CD8+ T cells in the bone marrow, and increase in expression of Tcf7, Slamf6, and Cxcr5 in CD8+ T cells. Finally, we profiled the epigenomes of in vivo JQ1-treated AML-derived CD8+ T cells by single-cell ATAC-seq and found that JQ1 increases Tcf7 accessibility specifically in Tex cells, suggesting that BETi likely acts mechanistically by relieving repression of progenitor programs in Tex CD8+ T cells and maintaining a pool of anti-PD1 responsive CD8+ T cells.
Summary Drug resistance in chronic myeloid leukaemia (CML) may occur via mutations in the causative BCR::ABL1 fusion or BCR::ABL1‐independent mechanisms. We analysed 48 patients with BCR::ABL1‐independent resistance for the presence of secondary fusion genes by RNA sequencing. We identified 10 of the most frequently detected secondary fusions in 21 patients. Validation studies, cell line models, gene expression analysis and drug screening revealed differences with respect to proliferation rate, differentiation and drug sensitivity. Notably, expression of RUNX1::MECOM led to resistance to ABL1 tyrosine kinase inhibitors in vitro. These results suggest secondary fusions contribute to BCR::ABL1‐independent resistance and may be amenable to combined therapies.
Acute myeloid leukemia (AML) is an aggressive myeloid lineage cancer with limited treatment options. Single-agent treatments with targeted inhibitors have resulted in some disease remissions, however, resistance almost always develops, prompting development of combinatorial treatment strategies. Studies have shown that AML patients exhibit markers of immune suppression such as increased levels of exhausted T cells (PD1/LAG3+) as well as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs)1. Inhibitors targeting the BET family, which are histone readers, have been shown to increase IFNy and IL-2 production in T cells by inhibiting BATF—a known negative regulator of TCF1, as well as decrease PD-L12–4. We, therefore, hypothesized that combining BRD4i and anti-PD1 would synergize by modulating both tumor intrinsic biology (though MYC reduction) and boosting T cell activity. We have characterized a FLT3-ITD/TET2/Lys-Cre mouse model of AML and observed significantly increased numbers of PD1+/TIM3+/TCF1− T cells, Tregs, and MDSCs when compared to healthy mice. T cells derived from mice with AML have significantly reduced T cell proliferation upon co-stimulation with CD3 and CD28. Ex vivo treatment with BRD4i + aPD1 significantly increased T cell proliferation in AML mice and increased TCF1 expression in a dose dependent manner. In vivo treatment with BRD4i + aPD1 showed enhanced reduction of leukemic blasts, reduced PD-L1, and increased CD8+ T cell infiltration into the bone marrow. Our work highlights the efficacy of this novel drug combination in targeting tumor intrinsic and extrinsic factors in AML and identifies a potential novel role for BRD4 in regulating TCF1 expression in T cells.
Acute myeloid leukemia (AML) is a hematological cancer with a very poor prognosis. FLT3-ITD mutations that cause constitutive FLT3 signaling are commonly seen in patients. Our lab and others have shown that AML patient samples exhibit hallmarks of immune exhaustion such as T cell dysfunction, increased MDSCs, and increased Tregs that associate with worse survival. In solid tumor models DCs have been shown to be important for anti-tumor T cell activity and we aim to further describe the role of DCs in the immune response to leukemia. FLT3 signaling is critical for DC development but the effects of FLT3-ITD on DCs and how it contributes to leukemogenesis remains unclear. In a novel FLT3-ITD driven AML mouse model we found that DCs from these mice have elevated pFLT3, indicating constitutive FLT3 signaling. Moreover, significantly expanded DC progenitors and DCs in the bone marrow and spleen are observed. Analysis of serum cytokines identified increased levels of IL-27, IL-10, and IL-17a in leukemia mice compared to healthy controls, suggesting that the expansion of DCs in these leukemia mice may be driving pathogenic expansion of T helper subsets. Furthermore, we measured higher levels of circulating Th2, Th17, and Treg phenotypes in the blood compared to healthy controls. Thus, we hypothesize that FLT3-ITD DCs are a significant contributor to T cell skewing in AML resulting in poor anti-tumor responses. We are using scRNA-seq methods to identify transcriptional changes in FLT3-ITD DCs that lead to the phenotypes we identified. Understanding how FLT3-ITD DCs contribute to AML immune suppression is critical to interpreting leukemia etiology and the development of targeted therapies such as immune checkpoint blockade or small molecule inhibitors.
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