RESUMEN
Richter's Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Linfoma de Células B Grandes Difuso , Animales , Ratones , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/patologíaRESUMEN
PURPOSE: Dual blockade of Bruton's tyrosine kinase with ibrutinib and selinexor has potential to deepen responses for patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: In this phase I study (clinicaltrials.gov: NCT02303392), adult patients with CLL/NHL, relapsed/refractory to ≥1 prior therapy were enrolled. Patients received weekly oral selinexor and daily oral ibrutinib in 28-day cycles until progression or intolerance. Primary objective was to determine MTD. RESULTS: Included patients had CLL (n = 16) or NHL (n = 18; 9 Richter transformation, 6 diffuse large B-cell lymphoma, and 3 mantle cell lymphoma). Median prior therapies were 4 (range = 1-14) and 59% previously received ibrutinib. The established MTD was 40 mg of selinexor (days 1, 8, 15) and 420 mg daily ibrutinib. Common nonhematologic adverse events were fatigue (56%), nausea (53%), anorexia (41%), and diarrhea (41%) and were mostly low grade. Overall response rate was 32%. An additional 47% achieved stable disease (SD), some prolonged (up to 36 months). Median progression-free survival for patients with CLL and NHL was 8.9 [95% confidence interval (CI), 3.9-16.1] and 2.7 (95% CI, 0.7-5.4) months, respectively. For patients with CLL who did not receive prior ibrutinib, only 20% (1/5) progressed. Estimated 2-year overall survival was 73.7% (95% CI, 44.1-89.2) and 27.8% (95% CI, 10.1-48.9) for patients with CLL and NHL, respectively. CONCLUSIONS: The selinexor and ibrutinib combination has demonstrated tolerability in patients with relapsed/refractory CLL/NHL. Responses were durable. Notable responses were seen in patients with CLL with minimal prior therapy. Future study of this combination will focus on efforts to deepen remissions in patients with CLL receiving ibrutinib therapy.
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Leucemia Linfocítica Crónica de Células B , Linfoma de Células B Grandes Difuso , Linfoma no Hodgkin , Adenina/análogos & derivados , Adulto , Humanos , Hidrazinas , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Piperidinas , Pirazoles/efectos adversos , Pirimidinas/efectos adversos , TriazolesRESUMEN
Using a genome-wide CRISPR screen, we identified CDK9, DHODH, and PRMT5 as synthetic lethal partners with gilteritinib treatment in fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) acute myeloid leukemia (AML) and genetically and pharmacologically validated their roles in gilteritinib sensitivity. The presence of FLT3-ITD is associated with an increase in anaerobic glycolysis, rendering leukemia cells highly sensitive to inhibition of glycolysis. Supportive of this, our data show the enrichment of single guide RNAs targeting 28 glycolysis-related genes upon gilteritinib treatment, suggesting that switching from glycolysis to oxidative phosphorylation (OXPHOS) may represent a metabolic adaption of AML in gilteritinib resistance. CDK9i/FLT3i, DHODHi/FLT3i, and PRMT5i/FLT3i pairs mechanistically converge on OXPHOS and purine biosynthesis blockade, implying that targeting the metabolic functions of these three genes and/or proteins may represent attractive strategies to sensitize AML to gilteritinib treatment. Our findings provide the basis for maximizing therapeutic impact of FLT3-ITD inhibitors and a rationale for a clinical trial of these novel combinations.
RESUMEN
Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.
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Biomarcadores de Tumor/genética , Evolución Clonal , Genómica/métodos , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , Mutación , Células Madre Neoplásicas/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo , Estudios de Seguimiento , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Pronóstico , Adulto JovenRESUMEN
Rare, recurrent balanced translocations occur in a variety of cancers but are often not functionally interrogated. Balanced translocations with the immunoglobulin heavy chain locus (IGH; 14q32) in chronic lymphocytic leukemia (CLL) are infrequent but have led to the discovery of pathogenic genes including CCND1, BCL2, and BCL3. Following identification of a t(X;14)(q28;q32) translocation that placed the mature T cell proliferation 1 gene (MTCP1) adjacent to the immunoglobulin locus in a CLL patient, we hypothesized that this gene may have previously unrecognized importance. Indeed, here we report overexpression of human MTCP1 restricted to the B cell compartment in mice produces a clonal CD5+/CD19+ leukemia recapitulating the major characteristics of human CLL and demonstrates favorable response to therapeutic intervention with ibrutinib. We reinforce the importance of genetic interrogation of rare, recurrent balanced translocations to identify cancer driving genes via the story of MTCP1 as a contributor to CLL pathogenesis.
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Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Translocación Genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Proteínas del Linfoma 3 de Células B , Ciclina D1 , Femenino , Regulación de la Expresión Génica , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Oncogenes/genética , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
BACKGROUND: Exportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood. METHODS: We performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor). RESULTS: We report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL. CONCLUSIONS: These findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.
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Regulación Leucémica de la Expresión Génica , Carioferinas/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Receptores Citoplasmáticos y Nucleares/genética , Animales , Epigénesis Genética , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , Estudios Retrospectivos , Transcriptoma , Proteína Exportina 1RESUMEN
Acute myeloid leukemia (AML) is a hematopoietic stem-cell-derived leukemia with often successive derived driver mutations. Late onset acquisition of internal tandem duplication in FLT3 (FLT3-ITD) at a high variant allele frequency often contributes to full transformation to a highly proliferative, rapidly progressive disease with poor outcome. The FLT3-ITD mutation is targetable with approved FLT3 small molecule inhibitors, including midostaurin and gilteritinib. However, outside of patients receiving allogeneic transplant, most patients fail to respond or relapse, suggesting alternative approaches of therapy will be required. We employed genome-wide pooled CRISPR knockout screening as a method for large-scale identification of targets whose knockout produces a phenotypic effect that enhances the antitumor properties of FLT3 inhibitors. Among the candidate targets we identified the effect of XPO1 knockout to be synergistic with midostaurin treatment. Next, we validated the genetic finding with pharmacologic combination of the slowly reversible XPO1 inhibitor selinexor with midostaurin and gilteritinib in FLT3-ITD AML cell lines and primary patient samples. Lastly, we demonstrated improved survival with either combination therapy compared to its monotherapy components in an aggressive AML murine model, supporting further evaluation and rapid clinical translation of this combination strategy.
RESUMEN
Acute myeloid leukemia (AML) is a heterogeneous and complex disease, and treatments for this disease have not been curative for the majority of patients. In younger patients, internal tandem duplication of FLT3 (FLT3-ITD) is a common mutation for which two inhibitors (midostaurin and gilteritinib) with varied potency and specificity for FLT3 are clinically approved. However, the high rate of relapse or failed initial response of AML patients suggests that the addition of a second targeted therapy may be necessary to improve efficacy. Using an unbiased large-scale CRISPR screen, we genetically identified BCL2 knockout as having synergistic effects with an approved FLT3 inhibitor. Here, we provide supportive studies that validate the therapeutic potential of the combination of FLT3 inhibitors with venetoclax in vitro and in vivo against multiple models of FLT3-ITD-driven AML. Our unbiased approach provides genetic validation for co-targeting FLT3 and BCL2 and repurposes CRISPR screening data, utilizing the genome-wide scope toward mechanistic understanding.