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1.
Mol Cancer Res ; 22(4): 386-401, 2024 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-38294692

RESUMO

Calcium homeostasis is critical for cell proliferation, and emerging evidence shows that cancer cells exhibit altered calcium signals to fulfill their need for proliferation. However, it remains unclear whether there are oncogene-specific calcium homeostasis regulations that can expose novel therapeutic targets. Here, from RNAi screen, we report that adenosylhomocysteinase like protein 1 (AHCYL1), a suppressor of the endoplasmic reticulum (ER) calcium channel protein inositol trisphosphate receptor (IP3R), is selectively upregulated and critical for cell proliferation and tumor growth potential of human NRAS-mutated melanoma, but not for melanoma expressing BRAF V600E. Mechanistically, AHCYL1 deficiency results in decreased ER calcium levels, activates the unfolded protein response (UPR), and triggers downstream apoptosis. In addition, we show that AHCYL1 transcription is regulated by activating transcription factor 2 (ATF2) in NRAS-mutated melanoma. Our work provides evidence for oncogene-specific calcium regulations and suggests AHCYL1 as a novel therapeutic target for RAS mutant-expressing human cancers, including melanoma. IMPLICATIONS: Our findings suggest that targeting the AHCYL1-IP3R axis presents a novel therapeutic approach for NRAS-mutated melanomas, with potential applicability to all cancers harboring RAS mutations, such as KRAS-mutated human colorectal cancers.


Assuntos
Adenosil-Homocisteinase , Retículo Endoplasmático , Melanoma , Humanos , Adenosil-Homocisteinase/metabolismo , Cálcio , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/genética , Homeostase , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo
2.
Blood ; 140(11): 1291-1304, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35763665

RESUMO

Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.


Assuntos
Calreticulina , Transtornos Mieloproliferativos , Animais , Calreticulina/genética , Calreticulina/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Glucose , Glicosilação , Humanos , Janus Quinase 2/genética , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Receptores de Trombopoetina/metabolismo
3.
Blood Cancer Discov ; 3(4): 298-315, 2022 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-35405004

RESUMO

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo. SIGNIFICANCE: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.


Assuntos
Calreticulina , Transtornos Mieloproliferativos , Neoplasias , Resposta a Proteínas não Dobradas , Cálcio/metabolismo , Calreticulina/genética , Endorribonucleases/genética , Humanos , Proteínas Mutantes/química , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a X-Box/genética
4.
Hematol Oncol ; 38(4): 554-559, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32583904

RESUMO

Ruxolitinib is effective in myeloproliferative neoplasms (MPN) but can cause reactivation of silent infections. We aimed at evaluating viral load and T-cell responses to human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in a cohort of 25 MPN patients treated with ruxolitinib. EBV-DNA and HCMV-DNA were quantified monthly using real-time polimerase chain reaction (PCR) on peripheral blood samples, and T-cell subsets were analyzed by flowcytometry. HCMV and EBV-directed T-cell responses were evaluated using the IFN-γ ELISPOT assay. Most patients had CD4+ and/or CD8+ T-cells below the normal range; these reductions were related to the duration of ruxolitinib treatment. In fact, reduced T-lymphocytes' subsets were found in 93% of patients treated for ≥5 years and in 45% of those treated for <5 years (P = .021). The former also had lower median numbers of CD4+ and CD8+ cells. Subclinical reactivation of EBV and HCMV occurred in 76% and 8% of patients. We observed a trend to an inverse relationship between EBV and CMV-specific CD4+ and CD8+ T-cell responses and viral load, and a trend to an inverse correlation with ruxolitinib dose. Therefore, our data suggest that the ruxolitinib treatment may interfere with immunosurveillance against EBV and HCMV.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Transtornos Mieloproliferativos/imunologia , Pirazóis/farmacologia , Ativação Viral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/virologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/imunologia , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/virologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/virologia , Feminino , Seguimentos , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/imunologia , Humanos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/virologia , Nitrilas , Prognóstico , Pirimidinas , Taxa de Sobrevida , Carga Viral , Ativação Viral/efeitos dos fármacos
6.
Hematology Am Soc Hematol Educ Program ; 2018(1): 110-117, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504299

RESUMO

Now that the spectrum of somatic mutations that initiate, propagate, and drive the progression of myeloproliferative neoplasms (MPNs) has largely been defined, recent efforts have focused on integrating this information into clinical decision making. In this regard, the greatest progress has been made in myelofibrosis, in which high-molecular-risk mutations have been identified and incorporated into prognostic models to help guide treatment decisions. In this chapter, we focus on advances in 4 main areas: (1) What are the MPN phenotypic driver mutations? (2) What constitutes high molecular risk in MPN (focusing on ASXL1)? (3) How do we risk-stratify patients with MPN? And (4) What is the significance of molecular genetics for MPN treatment? Although substantial progress has been made, we still have an incomplete understanding of the molecular basis for phenotypic diversity in MPN, and few rationally designed therapeutic approaches to target high-risk mutations are available. Ongoing research efforts in these areas are critical to understanding the biological consequences of genetic heterogeneity in MPN and to improving outcomes for patients.


Assuntos
Neoplasias Hematológicas , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/terapia , Prognóstico
7.
Oncotarget ; 8(60): 101735-101744, 2017 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-29254200

RESUMO

The recently revised World Health Organization (WHO) classification of myeloid neoplasms recognizes prefibrotic myelofibrosis (prePMF) as a distinct entity, characterized by well-defined histopathologic features together with minor clinical criteria (leukocytes, anemia, increased LDH, splenomegaly). The aim of the study was to examine the clinical relevance of distinguishing prePMF from essential thrombocythemia (ET). We identified in our database all patients affected with ET, prePMF and primary myelofibrosis (PMF) diagnosed according to 2008 WHO criteria with a bone marrow fibrosis grade 0-1 at diagnosis and one DNA sample to define the mutational status. The bone marrow morphology of all 404 identified patients was reviewed by an expert pathologist and patients were reclassified according to the 2016 WHO criteria. After reclassification, our cohort included 269 ET, 109 prePMF, and 26 myeloproliferative neoplasm unclassificable. In comparison with ET, patients with prePMF had higher leukocyte count, lower hemoglobin level, higher platelet count, higher LDH values, and higher number of circulating CD34-positive cells; they showed more frequently splenomegaly (all P values < ·001). CALR mutations were more frequent in prePMF than in ET (35·8% vs 17·8%, P < ·001). PrePMF patients had shorter overall survival (P < ·001) and a trend to a higher incidence of leukemic evolution (P ·067) compared to ET patients, while they did not differ in terms of thrombotic and bleeding complications. In conclusion, ET and prePMF diagnosed according to 2016 WHO criteria are two entities with a different clinical phenotype at diagnosis and a different clinical outcome.

8.
Oncotarget ; 8(20): 33416-33421, 2017 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-28422716

RESUMO

We investigated the variation of CALR-mutant burden during follow-up in 105 CALR-mutant MPN and compared it to the variation of JAK2-mutant burden in 226 JAK2-mutant MPN.The median allele burden at last evaluation was significantly higher than at first evaluation in essential thrombocythemia (ET) (49.5% vs 45%, P < .001) but not in primary myelofibrosis (PMF) (52% vs 51%, P 0.398). Median values of slope were positive both in ET (0.071) and in PMF (0.032). In CALR-mutant ET there was a difference between natural and therapy-related slope (P 0.006).In the JAK2-mutated cohort, the median allele burden at last evaluation was not different respect to that at first evaluation, neither in ET (22.9% vs 23.2%, P = 0.216) nor in PMF (50.5% vs 45.0%, P = 0.809), despite a positive slope. Multivariate analysis to evaluate the effect of mutation (CALR vs JAK2) on the slope of mutant burden in not treated pts with a positive slope adjusting for diagnosis (ET vs PMF) showed a trend toward a higher increase of mutant burden in CALR vs JAK2 (ß = 0.19, P = 0.061) with no difference between diagnosis (P = 0.419). The findings of this study suggest that clonal expansion in CALR-mutant MPN is faster than that observed in JAK2-mutant MPN.


Assuntos
Calreticulina/genética , Mutação , Transtornos Mieloproliferativos/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores , Análise Mutacional de DNA , Feminino , Seguimentos , Frequência do Gene , Genótipo , Humanos , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/diagnóstico , Prognóstico , Adulto Jovem
9.
Leuk Res ; 39(2): 236-41, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25498506

RESUMO

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterised by the clonal proliferation of the haematopoietic precursors together with the progressive development of bone marrow fibrosis. This stromal alteration is an important clinical issue and specific prognostic markers are not currently available. In bone marrow biopsies from 58 PMF patients, we explored the methylation pattern of genes encoding cytokines involved in the stromal reaction, namely platelet-derived growth factor-beta (PDGFB), transforming growth factor-beta (TGFB) and basic fibroblast growth factor (FGF2). We also evaluated the methylation profile of the Long Interspersed Nucleotide Element 1 (LINE-1). PDGFB, FGF2 and LINE-1, but not TGFB, were significantly differently methylated in PMF compared to controls. Significantly, PDGFB hypomethylation (<16%) was correlated with a favourable PMF prognosis (grade of marrow fibrosis, p=0.03; International Prognostic Scoring Systems p=0.01 and Dynamic International Prognostic Scoring Systems, p=0.02). Although the basis of the association of PDGFB hypomethylation with favourable prognosis remains to be clarified, we speculate that hypomethylation in PMF could represent the effect of acquired somatic mutations in genes involved in epigenetic regulation of the genome.


Assuntos
Metilação de DNA , Epigênese Genética , Mielofibrose Primária/metabolismo , Proteínas Proto-Oncogênicas c-sis/biossíntese , Biomarcadores/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos , Masculino , Pessoa de Meia-Idade , Mielofibrose Primária/genética , Prognóstico , Proteínas Proto-Oncogênicas c-sis/genética , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética
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