RESUMO
Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for a variety of hematological diseases. Allogenic HSCT requires hematopoietic stem cells (HSCs) from matched donors and comes with cytotoxicity and mortality. Recent advances in genome modification of HSCs have demonstrated the possibility of using autologous HSCT-based gene therapy to alleviate hematologic symptoms in monogenic diseases, such as the inherited bone marrow failure (BMF) syndrome Fanconi anemia (FA). However, for FA and other BMF syndromes, insufficient HSC numbers with functional defects results in delayed hematopoietic recovery and increased risk of graft failure. We and others previously identified the adaptor protein LNK (SH2B3) as a critical negative regulator of murine HSC homeostasis. However, whether LNK controls human HSCs has not been studied. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs results in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Importantly, LNK depletion enhances cytokine-mediated JAK/STAT activation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA. In xenotransplant, engraftment of FANCD2-depleted FA-like HSCs was markedly improved by LNK inhibition. Finally, targeting LNK in primary bone marrow HSPCs from FA patients enhanced their colony forming potential in vitro. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT and HSCT-based gene therapy.
Assuntos
Anemia de Fanconi , Transplante de Células-Tronco Hematopoéticas , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD34/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/terapia , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , CamundongosRESUMO
Internal tandem duplication within FLT3 (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and correlates with a poor prognosis. Whereas the FLT3 receptor tyrosine kinase is activated at the plasma membrane to transduce PI3K/AKT and RAS/MAPK signaling, FLT3-ITD resides in the endoplasmic reticulum and triggers constitutive STAT5 phosphorylation. Mechanisms underlying this aberrant FLT3-ITD subcellular localization or its impact on leukemogenesis remain poorly established. In this study, we discovered that FLT3-ITD is S-palmitoylated by the palmitoyl acyltransferase ZDHHC6. Disruption of palmitoylation redirected FLT3-ITD to the plasma membrane and rewired its downstream signaling by activating AKT and extracellular signal-regulated kinase pathways in addition to STAT5. Consequently, abrogation of palmitoylation increased FLT3-ITD-mediated progression of leukemia in xenotransplant-recipient mouse models. We further demonstrate that FLT3 proteins were palmitoylated in primary human AML cells. ZDHHC6-mediated palmitoylation restrained FLT3-ITD surface expression, signaling, and colonogenic growth of primary FLT3-ITD+ AML. More important, pharmacological inhibition of FLT3-ITD depalmitoylation synergized with the US Food and Drug Administration-approved FLT3 kinase inhibitor gilteritinib in abrogating the growth of primary FLT3-ITD+ AML cells. These findings provide novel insights into lipid-dependent compartmentalization of FLT3-ITD signaling in AML and suggest targeting depalmitoylation as a new therapeutic strategy to treat FLT3-ITD+ leukemias.
Assuntos
Leucemia/patologia , Lipoilação , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Duplicação Gênica , Humanos , Leucemia/genética , Leucemia/metabolismo , Camundongos SCID , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Inherited pathogenic variants in genes that confer moderate to high risk of breast cancer may explain up to 50% of familial breast cancer. This study aimed at identifying inherited pathogenic variants in breast cancer cases from Puerto Rico that were not linked to BRCA1 or BRCA2. Forty-eight breast cancer patients that met the clinical criteria for BRCA testing but had received a negative BRCA1/2 result were recruited. Fifty-three genes previously implicated in hereditary cancer predisposition were captured using the BROCA Agilent cancer risk panel followed by massively parallel sequencing. Missense variants of uncertain clinical significance in CHEK2 were evaluated using an in vitro kinase assays to determine their impact on function. Pathogenic variants were identified in CHEK2, MUTYH, and RAD51B in four breast cancer patients, which represented 8.3% of the cohort. We identified three rare missense variants of uncertain significance in CHEK2 and two variants (p.Pro484Leu and p.Glu239Lys) showed markedly decreased kinase activity in vitro comparable to a known pathogenic variant. Interestingly, the local ancestry at the RAD51B locus in the carrier of p.Arg47* was predicted to be of African origin. In this cohort, 12.5% of the BRCA-negative breast cancer patients were found to carry a known pathogenic variant or a variant affecting protein activity. This study reveals an unmet clinical need of genetic testing that could benefit a significant proportion of at-risk Latinas. It also highlights the complexity of Hispanic populations as pathogenic factors may originate from any of the ancestral populations that make up their genetic backgrounds.
Assuntos
Neoplasias da Mama/genética , Oncogenes , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/epidemiologia , Quinase do Ponto de Checagem 2/genética , DNA Glicosilases/genética , Proteínas de Ligação a DNA/genética , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Mutação Puntual , Porto Rico/epidemiologiaRESUMO
Prolonged exit from quiescence by hematopoietic stem cells (HSCs) progressively impairs their homeostasis in the bone marrow through an unidentified mechanism. We show that Rb proteins, which are major enforcers of quiescence, maintain HSC homeostasis by positively regulating thrombopoietin (Tpo)-mediated Jak2 signaling. Rb family protein inactivation triggers the progressive E2f-mediated transactivation of Socs3, a potent inhibitor of Jak2 signaling, in cycling HSCs. Aberrant activation of Socs3 impairs Tpo signaling and leads to impaired HSC homeostasis. Therefore, Rb proteins act as a central hub of quiescence and homeostasis by coordinating the regulation of both cell cycle and Jak2 signaling in HSCs.