Abstract Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of a subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.
Abstract Therapeutics targeted to specific proteins known to drive or modulate progression of acute myeloid leukemia (AML) have shown limited effectiveness as resistance frequently develops during their clinical use. Similar to many cancers, a stalled process of programmed cell death in apoptosis-primed cells is one of the prevalent mechanisms of drug resistance in AML. The intrinsic mitochondrial apoptotic pathway is controlled by a rheostat of anti-apoptotic and pro-apoptotic proteins and perturbing the balance toward pro-apoptotic responses remains a focus of many small molecular therapeutics. Venetoclax, an inhibitor of anti-apoptotic gene BCL2, approved for treatment of a certain subtypes of chronic lymphocytic leukemia (CLL), shows limited success as a monoagent in AML clinical trials due to an inherent resistance in approximately 37% of all patients (IC50 > 5μM). To identify mechanisms underlying intrinsic and acquired resistance to the BCL2 inhibitor, venetoclax, we used a genome-wide CRISPR/Cas9 screen to identify genes whose inactivation results in loss of venetoclax sensitivity in MOLM13 cells, derived from an AML patient that harbored mutation in FLT3-ITD, one of the major genetic landscapes of AML. Interrogation with two independently derived genome-wide libraries yielded identical top candidates TP53, BAX and PMAIP1. We also observed other gene targets that implicate the mitochondrial intrinsic, TP53-controlled pro-apoptotic pathway in the acquisition of venetoclax resistance. Correlatively, in a large AML cohort (Beat AML dataset), patients with loss of function mutations in TP53, or either low expression of TP53 or BAX exhibited statistically significant lower levels of responses to venetoclax ex vivo. Resultant venetoclax resistant cells with inactivation of TP53 or BAX had significantly reduced capacity for apoptosis when treated with venetoclax and exhibited elevated pro-survival signaling exemplified by increased levels of MAPK and AKT. In addition, loss of TP53 led to changes in the ratio of anti-apoptotic proteins BCL2, BCL-xL and MCL1 implicating TP53 as a major transcriptional regulator of the apoptotic rheostat in MOLM13 cells. Evaluation of the TP53 and BAX knockout cells for sensitivities to a panel of small molecule inhibitors revealed loss of sensitivities to a wide range of additional inhibitors including FLT3, AKT, multi-kinase inhibitors as well as a surprising gain of sensitivity to NTRK inhibitors relying on alternative pathways of cell death. Our results provide an independent confirmation of TP53 and its apoptotic network involvement to the venetoclax response in AML cells and suggest strategies to overcome resistance. Citation Format: Tamilla Nechiporuk, Stephen E. Kurtz, Olga Nikolova, Amanda d'Almeida, Kevin Watanabe-Smith, Mara Rosenberg, Brian Druker, Jeffrey W. Tyner, Shannon K. McWeeney. A genome-wide CRISPR screen on AML cells reveals the TP53 apoptotic network is a primary mediator of resistance to BCL2 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 325.
// Mara W. Rosenberg 1 , 2 , Kevin Watanabe-Smith 1 , 2 , Jeffrey W. Tyner 1 , 4 , Cristina E. Tognon 1 , 2 , 3 , Brian J. Druker 1 , 2 , 3 and Uma Borate 2 1 Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA 2 Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR, USA 3 Howard Hughes Medical Institute, Portland, OR, USA 4 Department of Cell, Developmental, and Cancer Biology, Oregon Health and Science University, Portland, OR, USA Correspondence to: Mara W. Rosenberg, email: rosenberg.mara@gmail.com Keywords: acute myeloid leukemia; drug sensitivity; midostaurin; drug resistance; FLT3 Abbreviations: AML: acute myeloid leukemia; FLT3-ITD: FLT3-internal tandem duplication; TKI: tyrosine kinase inhibitors; ELN Guidelines: European Leukemia Network Guidelines Received: April 23, 2020 Accepted: June 05, 2020 Published: July 21, 2020 ABSTRACT Acute myeloid leukemia (AML) is a heterogeneous malignancy with the most common genomic alterations in NPM1, DNMT3A, and FLT3. Midostaurin was the first FLT3 inhibitor FDA approved for AML and is standard of care for FLT3 mutant patients undergoing induction chemotherapy [ 1 , 2 ]. As there is a spectrum of response, we hypothesized that biological factors beyond FLT3 could play a role in drug sensitivity and that select FLT3-ITD negative samples may also demonstrate sensitivity. Thus, we aimed to identify features that would predict response to midostaurin in FLT3 mutant and wild-type samples. We performed an ex vivo drug sensitivity screen on primary and relapsed AML samples with corresponding targeted sequencing and RNA sequencing. We observed a correlation between FLT3-ITD mutations and midostaurin sensitivity as expected and observed KRAS and TP53 mutations correlating with midostaurin resistance in FLT3-ITD negative samples. Further, we identified genes differentially expressed in sensitive vs. resistant samples independent of FLT3-ITD status. Within FLT3-ITD mutant samples, over-expression of RGL4, oncogene and regulator of the Ras-Raf-MEK-ERK cascade, distinguished resistant from sensitive samples. Overall, this study highlights the complexity underlying midostaurin response. And, our results suggest that therapies that target both FLT3 and MAPK/ERK signaling may help circumvent some cases of resistance.
Acute myeloid leukemia is a poor prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and don’t always predict therapeutic response. Here we combine transcriptomic, proteomic, and phosphoproteomic datasets with ex vivo drug sensitivity data to help understand the underlying pathophysiology of AML beyond mutations. We measured the proteome and phosphoproteome of 210 patients and combined them with genomics and transcriptomic measurements to identify four proteogenomic subtypes that complemented existing genetic subtypes. We built a predictor to classify samples into subtypes and mapped them to a ‘landscape’ that identified specific drug response patterns, which predicted efficacious drug combinations. We then built a drug response prediction model to identify drugs that target distinct subtypes and validated our findings on cell lines representing various stages of quizartinib resistance. Our results show how multi-omics data together with drug sensitivity data can inform therapy stratification and drug combinations in AML.
<p>This file contains additional data such as detailed sequencing methods; Sanger sequencing result; receptor expression and function of R308 and double cysteine mutations.</p>
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