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T-prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm associated with marked chemotherapy resistance and continued poor clinical outcomes. Current treatments, i.e. the CD52-antibody alemtuzumab, offer transient responses, with relapses being almost inevitable without consolidating allogeneic transplantation. Recent more detailed concepts of T-PLL's pathobiology fostered the identification of actionable vulnerabilities: (i) altered epigenetics, (ii) defective DNA damage responses, (iii) aberrant cell-cycle regulation, and (iv) deregulated pro-survival pathways, including TCR and JAK/STAT signaling. To further develop related pre-clinical therapeutic concepts, we studied inhibitors of (H)DACs, BCL2, CDK, MDM2, and clas-sical cytostatics, utilizing (a) single-agent and combinatorial compound testing in 20 well-characterized and molecularly-profiled primary T-PLL (validated by additional 42 cases), and (b) 2 independent murine models (syngeneic transplants and patient-derived xenografts). Overall, the most efficient/selective single-agents and combinations (in vitro and in mice) in-cluded Cladribine, Romidepsin ((H)DAC), Venetoclax (BCL2), and/or Idasanutlin (MDM2). Cladribine sensitivity correlated with expression of its target RRM2. T-PLL cells revealed low overall apoptotic priming with heterogeneous dependencies on BCL2 proteins. In additional 38 T-cell leukemia/lymphoma lines, TP53 mutations were associated with resistance towards MDM2 inhibitors. P53 of T-PLL cells, predominantly in wild-type configuration, was amenable to MDM2 inhibition, which increased its MDM2-unbound fraction. This facilitated P53 activa-tion and down-stream signals (including enhanced accessibility of target-gene chromatin re-gions), in particular synergy with insults by Cladribine. Our data emphasize the therapeutic potential of pharmacologic strategies to reinstate P53-mediated apoptotic responses. The identified efficacies and their synergies provide an informative background on compound and patient selection for trial designs in T-PLL.
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Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.
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Leucemia Megacarioblástica Aguda , Leucemia Mieloide Aguda , Linfoma de Células B , Animais , Camundongos , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Proteína bcl-X/genética , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Diferenciação Celular , ApoptoseRESUMO
The BCL-2 inhibitor venetoclax has revolutionized the treatment of acute myeloid leukemia (AML) in patients not benefiting from intensive chemotherapy. Nevertheless, treatment failure remains a challenge, and predictive markers are needed, particularly for relapsed or refractory AML. Ex vivo drug sensitivity testing may correlate with outcomes, but its prospective predictive value remains unexplored. Here we report the results of the first stage of the prospective phase II VenEx trial evaluating the utility and predictiveness of venetoclax sensitivity testing using different cell culture conditions and cell viability assays in patients receiving venetoclax-azacitidine. Participants with de novo AML ineligible for intensive chemotherapy, relapsed or refractory AML, or secondary AML were included. The primary endpoint was the treatment response in participants showing ex vivo sensitivity and the key secondary endpoints were the correlation of sensitivity with responses and survival. Venetoclax sensitivity testing was successful in 38/39 participants. Experimental conditions significantly influenced the predictive accuracy. Blast-specific venetoclax sensitivity measured in conditioned medium most accurately correlated with treatment outcomes; 88% of sensitive participants achieved a treatment response. The median survival was significantly longer for participants who were ex vivo-sensitive to venetoclax (14.6 months for venetoclax-sensitive patients vs. 3.5 for venetoclax-insensitive patients, P<0.001). This analysis illustrates the feasibility of integrating drug-response profiling into clinical practice and demonstrates excellent predictivity. This trial is registered with ClinicalTrials.gov identifier: NCT04267081.
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Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Estudos Prospectivos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The presence of natural retinoids in vivo could influence the efficacy of pharmacologic agonists and antagonists. We found that natural RXRA ligands, but not RARA ligands, were present in murine MLL-AF9-derived myelomonocytic leukemias in vivo and that the concurrent presence of receptors and ligands acted as tumor suppressors. Pharmacologic retinoid responses could be optimized by concurrent targeting RXR ligands (e.g. bexarotene) and RARA ligands (e.g. all-trans retinoic acid, ATRA), which induced either leukemic maturation or apoptosis depending on cell culture conditions. Co-repressor release from the RARA:RXRA heterodimer occurred with RARA activation, but not RXRA activation, providing an explanation for the combination synergy. Combination synergy could be replicated in additional, but not all, AML cell lines and primary samples, and was associated with improved survival in vivo, although tolerability of bexarotene administration in mice remained an issue. These data provide insight into the basal presence of natural retinoids in leukemias in vivo and a potential strategy for clinical retinoid combination regimens in leukemias beyond acute promyelocytic leukemia.
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Leucemia Promielocítica Aguda , Retinoides , Animais , Diferenciação Celular , Camundongos , Receptores do Ácido Retinoico/genética , Tretinoína/farmacologiaRESUMO
MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5' end nonsense mutation (c.97G>T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin-α and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD.
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Códon sem Sentido , Complexo Mediador/genética , Degradação do RNAm Mediada por Códon sem Sentido , Motivos de Nucleotídeos , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Análise Mutacional de DNA , Humanos , Biossíntese de Proteínas , Transporte de RNARESUMO
The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.
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Agamaglobulinemia , Doenças Autoimunes , Doenças Genéticas Inatas , Leucemia Linfocítica Granular Grande , Mutação de Sentido Incorreto , Infecções por Mycobacterium , Fator de Transcrição STAT3 , Adolescente , Adulto , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/patologia , Substituição de Aminoácidos , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/imunologia , Leucemia Linfocítica Granular Grande/patologia , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/patologia , Estrutura Terciária de Proteína , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
BACKGROUND: T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS: We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS: Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS: The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).
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Leucemia Linfocítica Granular Grande/genética , Fator de Transcrição STAT3/genética , Idoso , Exoma , Expressão Gênica , Humanos , Masculino , Mutação , Receptores de Antígenos de Linfócitos T , Análise de Sequência de RNA , Transcrição Gênica , Regulação para CimaRESUMO
Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.
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Leucemia Linfocítica Granular Grande/genética , Fator de Transcrição STAT5/genética , Domínios de Homologia de src/genética , Idoso , Estudos de Coortes , Dimerização , Exoma/genética , Feminino , Testes Genéticos , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese , Mutação , Fosforilação/genética , Estrutura Terciária de Proteína , Fator de Transcrição STAT5/química , Fator de Transcrição STAT5/metabolismo , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genéticaAssuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Leucemia Mieloide Aguda , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Fenótipo , SulfonamidasRESUMO
CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of acute myeloid leukemia (AML) blasts, making it an attractive target for therapy of AML. Although previous CD33-targeting antibody-drug conjugates (ADC) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novelADCwith improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linkerpayloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated antitumor activity at single dose as low as 300 mg/kg in mice, while maintaining tolerability at single dose of 20 to 30 mg/kg in rats. In contrast with both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.
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Anticorpos Monoclonais Humanizados , Imunoconjugados , Leucemia Mieloide Aguda , Oligopeptídeos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Humanos , Animais , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Camundongos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Ratos , Anticorpos Monoclonais Humanizados/farmacologia , Oligopeptídeos/farmacologia , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Aminobenzoatos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , FemininoRESUMO
The principle of drug sensitivity testing is to expose cancer cells to a library of different drugs and measure its effects on cell viability. Recent technological advances, continuous approval of targeted therapies, and improved cell culture protocols have enhanced the precision and clinical relevance of such screens. Indeed, drug sensitivity testing has proven diagnostically valuable for patients with advanced hematologic cancers. However, different cell types behave differently in culture and therefore require optimized drug screening protocols to ensure that their ex vivo drug sensitivity accurately reflects in vivo drug responses. For example, primary chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells require unique microenvironmental stimuli to survive in culture, while this is less the case for acute myeloid leukemia (AML) cells. Here, we present our optimized and validated protocols for culturing and drug screening of primary cells from AML, CLL, and MM patients, and a generic protocol for cell line models. We also discuss drug library designs, reproducibility, and quality controls. We envision that these protocols may serve as community guidelines for the use and interpretation of assays to monitor drug sensitivity in hematologic cancers and thus contribute to standardization. The read-outs may provide insight into tumor biology, identify or confirm treatment resistance and sensitivity in real time, and ultimately guide clinical decision-making.
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We generated ex vivo drug-response and multiomics profiling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identification of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profiling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes significant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275.
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Técnicas de Apoio para a Decisão , Leucemia Mieloide Aguda/tratamento farmacológico , Equipe de Assistência ao Paciente , Medicina de Precisão , Feminino , Finlândia , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Análise de SobrevidaRESUMO
Mature natural killer (NK) cell neoplasms are rare but very aggressive types of cancers. With currently available treatments, they have a very poor prognosis and, as such, are an example of group of cancers in which the development of effective precision therapies is needed. Using both short- and long-term drug sensitivity testing, we explored novel ways to target NK-cell neoplasms by combining the clinically approved JAK inhibitor ruxolitinib with other targeted agents. We profiled 7 malignant NK-cell lines in drug sensitivity screens and identified that these exhibit differential drug sensitivities based on their genetic background. In short-term assays, various classes of drugs combined with ruxolitinib seemed highly potent. Strikingly, resistance to most of these combinations emerged rapidly when explored in long-term assays. However, 4 combinations were identified that selectively eradicated the cancer cells and did not allow for development of resistance: ruxolitinib combined with the mouse double-minute 2 homolog (MDM2) inhibitor idasanutlin in STAT3-mutant, TP53 wild-type cell lines; ruxolitinib combined with the farnesyltransferase inhibitor tipifarnib in TP53-mutant cell lines; and ruxolitinib combined with either the glucocorticoid dexamethasone or the myeloid cell leukemia-1 (MCL-1) inhibitor S63845 but both without a clear link to underlying genetic features. In conclusion, using a new drug sensitivity screening approach, we identified drug combinations that selectively target mature NK-cell neoplasms and do not allow for development of resistance, some of which can be applied in a genetically stratified manner.
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Antineoplásicos , Neoplasias , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Combinação de Medicamentos , Células Matadoras Naturais , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
The FDA recently approved eight targeted therapies for acute myeloid leukemia (AML), including the BCL-2 inhibitor venetoclax. Maximizing efficacy of these treatments requires refining patient selection. To this end, we analyzed two recent AML studies profiling the gene expression and ex vivo drug response of primary patient samples. We find that ex vivo samples often exhibit a general sensitivity to (any) drug exposure, independent of drug target. We observe that this "general response across drugs" (GRD) is associated with FLT3-ITD mutations, clinical response to standard induction chemotherapy, and overall survival. Further, incorporating GRD into expression-based regression models trained on one of the studies improved their performance in predicting ex vivo response in the second study, thus signifying its relevance to precision oncology efforts. We find that venetoclax response is independent of GRD but instead show that it is linked to expression of monocyte-associated genes by developing and applying a multi-source Bayesian regression approach. The method shares information across studies to robustly identify biomarkers of drug response and is broadly applicable in integrative analyses.
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The extensive drug resistance requires rational approaches to design personalized combinatorial treatments that exploit patient-specific therapeutic vulnerabilities to selectively target disease-driving cell subpopulations. To solve the combinatorial explosion challenge, we implemented an effective machine learning approach that prioritizes patient-customized drug combinations with a desired synergy-efficacy-toxicity balance by combining single-cell RNA sequencing with ex vivo single-agent testing in scarce patient-derived primary cells. When applied to two diagnostic and two refractory acute myeloid leukemia (AML) patient cases, each with a different genetic background, we accurately predicted patient-specific combinations that not only resulted in synergistic cancer cell co-inhibition but also were capable of targeting specific AML cell subpopulations that emerge in differing stages of disease pathogenesis or treatment regimens. Our functional precision oncology approach provides an unbiased means for systematic identification of personalized combinatorial regimens that selectively co-inhibit leukemic cells while avoiding inhibition of nonmalignant cells, thereby increasing their likelihood for clinical translation.
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STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers.
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Antineoplásicos/farmacologia , Biologia Computacional , Fator de Transcrição STAT3/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Fatores de TempoRESUMO
Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.
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Janus Quinases/genética , Leucemia Linfocítica Granular Grande/genética , Mutação , Fator de Transcrição STAT3/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Humanos , Janus Quinases/metabolismo , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/terapia , Masculino , Pessoa de Meia-Idade , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sequenciamento do Exoma , Adulto JovemRESUMO
Novel agents have increased survival of multiple myeloma (MM) patients, however high-risk and relapsed/refractory patients remain challenging to treat and their outcome is poor. To identify novel therapies and aid treatment selection for MM, we assessed the ex vivo sensitivity of 50 MM patient samples to 308 approved and investigational drugs. With the results we i) classified patients based on their ex vivo drug response profile; ii) identified and matched potential drug candidates to recurrent cytogenetic alterations; and iii) correlated ex vivo drug sensitivity to patient outcome. Based on their drug sensitivity profiles, MM patients were stratified into four distinct subgroups with varied survival outcomes. Patients with progressive disease and poor survival clustered in a drug response group exhibiting high sensitivity to signal transduction inhibitors. Del(17p) positive samples were resistant to most drugs tested with the exception of histone deacetylase and BCL2 inhibitors. Samples positive for t(4;14) were highly sensitive to immunomodulatory drugs, proteasome inhibitors and several targeted drugs. Three patients treated based on the ex vivo results showed good response to the selected treatments. Our results demonstrate that ex vivo drug testing may potentially be applied to optimize treatment selection and achieve therapeutic benefit for relapsed/refractory MM.
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Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function STAT3 mutations in lymphoid cancers leading to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3.
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Tyrosine kinase inhibitors (TKI) are the mainstay treatment of BCR-ABL1-positive leukemia and virtually all patients with chronic myeloid leukemia in chronic phase (CP CML) respond to TKI therapy. However, there is limited information on the cellular mechanisms of response and particularly on the effect of cell differentiation state to TKI sensitivity in vivo and ex vivo/in vitro. We used multiple, independent high-throughput drug sensitivity and resistance testing platforms that collectively evaluated 295 oncology compounds to characterize ex vivo drug response profiles of primary cells freshly collected from newly-diagnosed patients with BCR-ABL1-positive leukemia (n = 40) and healthy controls (n = 12). In contrast to the highly TKI-sensitive cells from blast phase CML and Philadelphia chromosome-positive acute lymphoblastic leukemia, primary CP CML cells were insensitive to TKI therapy ex vivo. Despite maintaining potent BCR-ABL1 inhibitory activity, ex vivo viability of cells was unaffected by TKIs. These findings were validated in two independent patient cohorts and analysis platforms. All CP CML patients under study responded to TKI therapy in vivo. When CP CML cells were sorted based on CD34 expression, the CD34-positive progenitor cells showed good sensitivity to TKIs, whereas the more mature CD34-negative cells were markedly less sensitive. Thus in CP CML, TKIs predominantly target the progenitor cell population while the differentiated leukemic cells (mostly cells from granulocytic series) are insensitive to BCR-ABL1 inhibition. These findings have implications for drug discovery in CP CML and indicate a fundamental biological difference between CP CML and advanced forms of BCR-ABL1-positive leukemia.