RESUMEN
Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Antineoplásicos/uso terapéutico , Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Ceramidas/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.
Asunto(s)
Calreticulina , Trastornos Mieloproliferativos , Animales , Anticuerpos Monoclonales , Calreticulina/genética , Calreticulina/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , RatasRESUMEN
Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.
RESUMEN
Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax. SIGNIFICANCE: Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions. See related commentary by Robinson and Levine, p. 266. This article is highlighted in the In This Issue feature, p. 247.