performed experiments and analyzed data. Qinghai Ye provided patient tissue samples. Qiongzhu Dong, Hirohito Yamaguchi, and Yi Du provided scientific input and critical cells. Jennifer L. Hsu revised the manuscript. Mien-Chie Hung supervised the entire project, designed the experiments, analyzed data, and wrote the manuscript.

Concomitant targeting of BCL2 with venetoclax and MAPK signaling with cobimetinib in acute myeloid leukemia models. Abstract:The pathogenesis of acute myeloid leukemia involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes to effectively treat the disease. The BCL2 selective inhibitor venetoclax has potent antileukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of BCL2 and MAPK pathways by venetoclax and MEK1/2 inhibitor cobimetinib respectively. The combination demonstrated synergy in 7 of 11 acute myeloid leukemia cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary patient samples with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo, the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in acute myeloid leukemia. 5 Methods Patient samples, AML cell lines, and reagentsBone marrow and peripheral blood samples were collected from patients with AML or healthy donors after informed consent was obtained in accordance with the Institutional Review Board of The University of Texas MD Anderson Cancer Center. The cell line culture methodology is described in the Supplemental Methods. CellTiter-Glo proliferation assayDetails are provided in the Supplemental Methods. Apoptosis in primary AML samplesAs previously reported, 24 18 primary AML peripheral blood mononuclear cells or AML PDX samples were cultured in LSC medium. Viable AML CD45 dim blast cells were enumerated by using CountBright counting beads (Cat. C36950; Invitrogen, Carlsbad, CA) with concurrent Annexin-V and DAPI detection on a Gallios Flow Cytometer (Beckman Coulter, Indianapolis, IN). Data analysis and additional details are included in Supplemental Methods. Colony-forming cell assay Details are provided in the Supplemental Methods. Electrochemiluminescent ELISA assay, reverse-phase protein array, and RNA sequencing Details are provided in...

FMS‐like tyrosine kinase 3 (FLT3) mutations are prevalent in acute myeloid leukemia (AML), and their presence confers adverse risk. FLT3‐mutated (FLT3m) AML is a challenging leukemia to manage, particularly in older and unfit patients as well as patients with relapsed/refractory (r/r) disease. We retrospectively analyzed the outcomes of 50 FLT3m AML patients (17 treatment‐naïve, 33 r/r) treated with venetoclax (VEN) and hypomethylating agents (HMA). The overall CR/CRi rate with VEN‐HMA was 60% (94% in treatment‐naïve AML and 42% in r/r AML). Early (60‐days) treatment related mortality was 2%. The r/r AML setting was an independent predictor of lower complete response (OR: 0.08; 95%CI: 0.00‐0.60, P = .03). Cytogenetics‐molecular risk, concurrent mutations, the type of FLT3 mutation (ITD vs TKD), the ITD allelic ratio, the type of HMA, age, prior exposure to HMA and receipt of prior allogeneic transplant did not independently impact response or leukemia‐free survival (LFS). Concurrent IDH mutations were associated with lower CR/CRi (P = .01), while ASXL1 or TET2 mutations showed a non‐significant association toward higher CR/CRi (P = .07, for both). However, none of the concurrent mutations were an independent predictor for response when adjusted to AML setting. In conclusion, VEN‐HMA is associated with encouraging efficacy in FLT3m AML among both newly diagnosed unfit and r/r patients.

BACKGROUND: Blinatumomab has demonstrated encouraging activity in relapsed/refractory (r/r) and minimal residual disease-positive (MRD+) acute lymphoblastic leukemia (ALL). Extramedullary disease (EMD) relapse or relapse with CD19-disease has been observed after blinatumomab therapy in patients with r/r or MRD+ ALL. However, the pathophysiology and risk factors of treatment failure are not fully understood. METHODS: This study retrospectively reviewed the outcomes of adult patients with B-cell ALL treated with blinatumomab (n = 132) for either r/r (n = 103) or MRD+ disease (n = 29) at the authors' center (2013-2021) and analyzed factors associated with treatment response and EMD failure. RESULTS: The overall response rate was 64%. A lower marrow blast burden before blinatumomab (P = .049) and no history of previous EMD (P = .019) were significantly associated with a higher response. Among the patients who responded to blinatumomab, 56% underwent consolidation with allogeneic transplantation. Blinatumomab failure was observed in 89 patients; 43% of these patients (n = 38) either progressed or relapsed at extramedullary sites. A history of extramedullary involvement (53% vs 24%; P = .005) and retention of CD19 expression at the time of relapse/progression (97% vs 74%; P = .012) were associated with a higher risk for extramedullary failure. Central nervous system (CNS) failure after blinatumomab was encountered in 39% of the patients with EMD. CONCLUSIONS: A history of EMD predicted an inferior response to blinatumomab therapy with a higher risk for relapse/ progression at extramedullary sites (particularly CNS). Consolidation with allogenic transplantation in patients who primarily responded to blinatumomab did not abrogate the risk of extramedullary relapse. The incorporation of extramedullary assessment and the intensification of CNS prophylaxis may help in addressing extramedullary failure.

Two articles in this week’s issue focus on the use of ipilimumab and decitabine for patients with myelodysplasia (MDS) and acute myeloid leukemia (AML) before and after hematopoietic stem cell transplantation (HSCT) for high-risk disease. In the first article, Garcia et al report on the results of a phase 1 trial of the combination in 54 patients, demonstrating overall response rate of 52% in patients who are HSCT-naïve and 20% in patients post-HSCT; responses are usually short-lived. In the second article, Penter and colleagues characterize gene expression responses to therapy and conclude that decitabine acts directly to clear leukemic cells while ipilimumab acts on infiltrating lymphocytes in marrow and extramedullary sites. Responses are determined by leukemic cell burden and by the frequency and phenotype of infiltrating lymphocytes. Increasing bone marrow regulatory T cells is identified as a potential contributor to checkpoint inhibitor escape.