RESUMEN
In many cancers, mortality is associated with the emergence of relapse with multidrug resistance (MDR). Thus far, the investigation of cancer relapse mechanisms has largely focused on acquired genetic mutations. Using acute myeloid leukemia (AML) patient-derived xenografts (PDX), we systematically elucidated a basis of MDR and identified drug sensitivity in relapsed AML. We derived pharmacologic sensitivity for 22 AML PDX models using dynamic BH3 profiling (DBP), together with genomics and transcriptomics. Using in vivo acquired resistant PDXs, we found that resistance to unrelated, narrowly targeted agents in distinct PDXs was accompanied by broad resistance to drugs with disparate mechanisms. Moreover, baseline mitochondrial apoptotic priming was consistently reduced regardless of the class of drug-inducing selection. By applying DBP, we identified drugs showing effective in vivo activity in resistant models. This study implies evasion of apoptosis drives drug resistance and demonstrates the feasibility of the DBP approach to identify active drugs for patients with relapsed AML. SIGNIFICANCE: Acquired resistance to targeted therapy remains challenging in AML. We found that reduction in mitochondrial priming and common transcriptomic signatures was a conserved mechanism of acquired resistance across different drug classes in vivo. Drugs active in vivo can be identified even in the multidrug resistant state by DBP.
Asunto(s)
Apoptosis , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/genética , Humanos , Apoptosis/efectos de los fármacos , Animales , Ratones , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Resistencia a Múltiples Medicamentos/genética , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Células Precursoras de Granulocitos/efectos de los fármacos , Células Precursoras de Granulocitos/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies, including multiple myeloma (MM). However, this plasma cell malignancy remains incurable, and novel therapies are therefore urgently needed. Whole-genome transcriptome analyses in a large cohort of MM patients demonstrated that alterations in pre-mRNA splicing (AS) are frequent in MM. This manuscript describes approaches to identify disease-specific alterations in MM and proposes RNA-based therapeutic strategies to eradicate such alterations. As a "proof of concept", we examined the causes of aberrant HMMR (Hyaluronan-mediated motility receptor) splicing in MM. We identified clusters of single nucleotide variations (SNVs) in the HMMR transcript where the altered splicing took place. Using bioinformatics tools, we predicted SNVs and splicing factors that potentially contribute to aberrant HMMR splicing. Based on bioinformatic analyses and validation studies, we provided the rationale for RNA-based therapeutic strategies to selectively inhibit altered HMMR splicing in MM. Since splicing is a hallmark of many cancers, strategies described herein for target identification and the design of RNA-based therapeutics that inhibit gene splicing can be applied not only to other genes in MM but also more broadly to other hematological malignancies and solid tumors as well.
Asunto(s)
Neoplasias Hematológicas , Mieloma Múltiple , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Empalme Alternativo , ARN , Empalme del ARNRESUMEN
Oncogenic activated RAS mutations have been detected in 50% of de novo and 70% of relapsed multiple myeloma (MM) patients. Translocation t(11;14) involving IgH/CCDN1 and overexpression of cyclin-Ds are early events in MM pathogenesis, enhancing uncontrolled MM cell growth. We hypothesized that targeting both RAS/MAPK pathway molecules including Erk1/2 along with cyclin-Ds enhances MM cytotoxicity and minimizes side effects. Recent studies have demonstrated the high potency of Erk1/2 and CDK4/6 inhibitors in metastatic relapsed cancers, and here we tested anti-MM effects of the Erk1/2 + CDK4/6 inhibitor combination. Our studies showed strong synergistic (IC < 0.5) cytotoxicity of Erk1/2i + CDK4/6i in MM-cells. Erk1/2i + CDK4/6i treatment in a dose-dependent manner arrested MM-cells in the G0/G1 phase and activated mitochondrial apoptotic signaling. Our studies showed that Erk1/2i + CDK4/6i treatment-induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling, such as c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of these inhibitors. We identified Erk1/2i + CDK4/6i treatment associated five-gene signature which includes SNRPB and SLC25A5; these genes are involved in RNA processing and mitochondrial metabolism, respectively. Overall, our studies provide the preclinical framework for Erk1/2i + CDK4/6i combination clinical trials to target Ras+CDK pathways to improve patient outcome in MM.
Asunto(s)
Neoplasias de la Mama , Mieloma Múltiple , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Femenino , Humanos , Mieloma Múltiple/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/patología , Mitocondrias/metabolismo , Pirimidinas/farmacología , Sulfonamidas/farmacología , Tiofenos/farmacología , Animales , Apoptosis , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas/farmacología , Transducción de SeñalRESUMEN
Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Quinasas Janus/metabolismo , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/metabolismo , Factores de Transcripción STAT/metabolismo , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Médula Ósea/metabolismo , Médula Ósea/patología , Humanos , Quinasas Janus/efectos de los fármacos , Mieloma Múltiple/patología , Nitrilos , Pirazoles/farmacología , Pirimidinas , Factores de Transcripción STAT/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
BACKGROUND: There is growing evidence that spleen tyrosine kinase (SYK) is critical for acute myeloid leukaemia (AML) transformation and maintenance of the leukemic clone in AML patients. It has also been found to be over-expressed in AML patients, with activating mutations in foetal liver tyrosine kinase 3 (FLT3), particularly those with internal tandem duplications (FLT3-ITD), where it transactivates FLT3-ITD and confers resistance to treatment with FLT3 tyrosine kinase inhibitors (TKIs). METHODS: We have previously described a pharmacological approach to treating FLT3-ITD-positive AML that relies on proteasome-mediated FLT3 degradation via inhibition of USP10, the deubiquitinating enzyme (DUB) responsible for cleaving ubiquitin from FLT3. RESULTS: Here, we show that USP10 is also a major DUB required for stabilisation of SYK. We further demonstrate that degradation of SYK can be induced by USP10-targeting inhibitors. USP10 inhibition leads to death of cells driven by active SYK or oncogenic FLT3 and potentiates the anti-leukemic effects of FLT3 inhibition in these cells. CONCLUSIONS: We suggest that USP10 inhibition is a novel approach to inhibiting SYK and impeding its role in the pathology of AML, including oncogenic FLT3-positive AML. Also, given the significant transforming role SYK in other tumours, targeting USP10 may have broader applications in cancer.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Quinasa Syk/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Células Cultivadas , Humanos , Quinasa Syk/antagonistas & inhibidores , Ubiquitina Tiolesterasa/fisiología , Ubiquitinación , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine-independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non-receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3-positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL-positive leukaemia. Taken together, our results support the notion that mutant CBL-expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.
Asunto(s)
Leucemia Mieloide/genética , Mutación/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Quinasa Syk/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Línea Celular Tumoral , Humanos , Leucemia Mieloide/tratamiento farmacológico , Ratones , Mutación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Estaurosporina/análogos & derivados , Estaurosporina/farmacologíaRESUMEN
Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA-approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi-targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild-type (wt) FLT3-expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.
Asunto(s)
Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Estaurosporina/análogos & derivados , Tirosina Quinasa 3 Similar a fms/genética , Compuestos de Anilina/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bencimidazoles/farmacología , Benzotiazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Mutación/efectos de los fármacos , Compuestos de Fenilurea/farmacología , Piperidinas/farmacología , Pirazinas/farmacología , Sorafenib/farmacología , Estaurosporina/farmacología , Quinasa Syk/genética , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
Over the last few years, a detailed map of genetic and epigenetic lesions that underlie multiple myeloma (MM) has been created. Regulation of microRNA (miR)-dependent gene expression and mRNA splicing play significant roles in MM pathogenesis; however, to date an interplay between these processes is not yet delineated. Here we investigated miR-mediated regulation of splicing networks at the transcriptome level. Our studies show that a significant number (78%) of miRs which are either up- or down-regulated in patient CD138+ MM cells, but not in healthy donors (HD) CD138+ plasma cells (PC), target genes involved in early stages of pre-mRNA splicing. We also identified deregulated miRs that target core splicing factors (SF) and modifiers (SM, enhancers/silencers) which cause altered splicing in MM. Our studies suggest that Let-7f, in combination other miRs which are frequently and significantly deregulated in patients with overt MM, targets genes that regulate intron excision. Importantly, deregulated expression of certain miRs in MM promote increased intron retention, a novel characteristic of the MM genome, by inducing deregulated expression of the genes that regulate the splicing network. Our studies, therefore, provide the rationale for therapeutically targeting deregulated miRs to reverse aberrant splicing and improve patient outcome in MM.
Asunto(s)
MicroARNs/genética , Mieloma Múltiple/genética , Precursores del ARN/genética , Empalme del ARN/genética , HumanosRESUMEN
Mutations in two type-3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3-positive AML and for KIT D816V-positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild-type (wt) FLT3, FLT3-internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3-ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c-CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , Proteínas Mutantes/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Antineoplásicos/uso terapéutico , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Benzotiazoles/farmacología , Benzotiazoles/uso terapéutico , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Hematológicas/genética , Humanos , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Piperidinas/farmacología , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-cbl/efectos de los fármacos , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Proto-Oncogénicas c-kit/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/genética , Pirazinas/farmacología , Pirazinas/uso terapéutico , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirroles/farmacología , Pirroles/uso terapéutico , Sorafenib/farmacología , Sorafenib/uso terapéutico , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Estaurosporina/uso terapéutico , Triazinas/farmacología , Triazinas/uso terapéutico , Tirosina Quinasa 3 Similar a fms/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) cells harboring mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) produce the oncometabolite 2-hydroxyglutarate (2HG). This study prospectively evaluated the 2HG levels, IDH1/2 mutational status, and outcomes of patients receiving standard chemotherapy for newly diagnosed AML. METHODS: Serial samples of serum, urine, and bone marrow aspirates were collected from patients newly diagnosed with AML, and 2HG levels were measured with mass spectrometry. Patients with baseline serum 2HG levels greater than 1000 ng/mL or marrow pellet 2HG levels greater than 1000 ng/2 × 106 cells, which suggested the presence of an IDH1/2 mutation, underwent serial testing. IDH1/2 mutations and estimated variant allele frequencies were identified. AML characteristics were compared with the Wilcoxon test and Fisher's exact test. Disease-free survival and overall survival (OS) were evaluated with log-rank tests and Cox regression. RESULTS: Two hundred and two patients were treated for AML; 51 harbored IDH1/2 mutations. IDH1/2-mutated patients had significantly higher 2HG levels in serum, urine, bone marrow aspirates, and aspirate cell pellets than wild-type patients. A serum 2HG level greater than 534.5 ng/mL was 98.8% specific for the presence of an IDH1/2 mutation. Patients with IDH1/2-mutated AML treated with 7+3-based induction had a 2-year event-free survival (EFS) rate of 44% and a 2-year OS rate of 57%. There was no difference in complete remission rates, EFS, or OS between IDH1/2-mutated and wild-type patients. Decreased serum 2HG levels on day 14 as a proportion of the baseline were significantly associated with improvements in EFS (P = .047) and OS (P = .019) in a multivariate analysis. CONCLUSIONS: Among patients with IDH1/2-mutated AML, 2HG levels are highly specific for the mutational status at diagnosis, and they have prognostic relevance in patients receiving standard chemotherapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Glutaratos/sangre , Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/mortalidad , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia , Adulto JovenRESUMEN
Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Tiofenos/farmacología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Estructura Molecular , Mutación , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Tiofenos/química , Células Tumorales Cultivadas , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
PI3Kδ is predominately expressed in leukocytes and has been found overexpressed in B-cell related malignances such as CLL and AML. We have discovered a highly selective ATP competitive PI3Kd inhibitor PI3KD-IN-015, which exhibits a high selectivity among other PI3K isoforms in both biochemical assays and cellular assay, meanwhile did not inhibit most of other protein kinases in the kinome. PI3KD-IN-015 demonstrates moderately anti-proliferation efficacies against a variety of B-cell related cancer cell lines through down-regulate the PI3K signaling significantly. It induced both apoptosis and autophagy in B-cell malignant cell lines. In addition, combination of autophagy inhibitor Bafilomycin could potentiate the moderate anti-proliferation effect of PI3KD-IN-015. PI3KD-IN-015 shows anti-proliferation efficacy against CLL and AML patient primary cells. Collectively, these results indicate that PI3KD-IN-015 may be useful drug candidate for further development of anti-B-cell related malignances therapies.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Leucemia de Células B/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Humanos , Leucemia de Células B/enzimología , Leucemia de Células B/patología , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/químicaRESUMEN
Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.
Asunto(s)
Daño del ADN , Mieloma Múltiple/enzimología , Mieloma Múltiple/patología , Sirtuinas/metabolismo , Acetilación , Línea Celular Tumoral , Proliferación Celular , Reparación del ADN , Doxorrubicina/farmacología , Histonas/metabolismo , Humanos , Lisina/metabolismo , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Mutágenos/toxicidad , Pronóstico , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
PURPOSE: The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have antileukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current antileukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here, we evaluated the antileukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. EXPERIMENTAL DESIGN: The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n = 6; B-CLL, n = 19), and healthy leukocytes. Intracellular nicotinamide adenine dinucleotide (NAD(+)) and ATP levels, mitochondrial transmembrane potential (ΔΨ(m)), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. RESULTS: The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34(+) progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD(+) and ATP shortage, and induced ΔΨ(m) dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. CONCLUSIONS: APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including acute myelogenous leukemia (AML) and B-cell chronic lymphocytic leukemia (B-CLL) samples. Further evaluations of the combination of these agents in clinical setting should be considered.
Asunto(s)
Acrilamidas/farmacología , Antineoplásicos/farmacología , Ciclosporina/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Leucemia/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Piperidinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Anciano , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Aberraciones Cromosómicas , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Expresión Génica , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia/genética , Leucemia/mortalidad , Leucemia/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Mutación , NAD/metabolismo , Estadificación de Neoplasias , Niacina/farmacología , Niacinamida/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Cultivo Primario de Células , Pronóstico , Células Tumorales Cultivadas , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
It is becoming increasingly apparent that splicing defects play a key role in cancer, and that alterations in genomic splicing elements promote aberrant splicing. Alternative splicing increases the diversity of the human transcriptome and increases the numbers of functional gene products. However, dysregulation that leads to aberrant pre-mRNA splicing can contribute to cancer. Hyaluronan (HA), known to be an important component of cancer progression, is synthesized by hyaluronan synthases (HASs). In cancer cells, hyaluronan synthase 1 (HAS1) pre-mRNA is abnormally spliced to generate a family of aberrant splice variants (HAS1Vs) that synthesize extracellular and intracellular HA. HAS1Vs are clinically relevant, being found almost exclusively in malignant cells. Expression of aberrant HAS1Vs predicts poor survival in multiple myeloma. In this review, we summarize the unusual properties of HAS1Vs and their relationship to cancer. HAS1Vs form heterogeneous multimers with normally spliced HAS1 as well as with each other and with HAS3. Aberrant variants of HAS1 synthesize HA. Extracellular HA synthesized by HAS1Vs is likely to promote malignant spread. We speculate that synthesis of intracellular HA plays a fundamental and early role in oncogenesis by promoting genetic instability and the emergence of viable cancer variants that lead to aggressive disease.
Asunto(s)
Transformación Celular Neoplásica , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Neoplasias/metabolismo , Procesamiento Postranscripcional del ARN , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Progresión de la Enfermedad , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Variación Genética , Proteínas Fluorescentes Verdes/química , Humanos , Hialuronano Sintasas , Ratones , Mitosis , Datos de Secuencia Molecular , Mieloma Múltiple/patología , Mutación , Neoplasias/genética , ARN Mensajero/metabolismo , Huso Acromático , Resultado del TratamientoRESUMEN
Genetic variations in the hyaluronan synthase 1 gene (HAS1) influence HAS1 aberrant splicing. HAS1 is aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM), but not in their counterparts from healthy donors. The presence of aberrant HAS1 splice variants predicts for poor survival in multiple myeloma (MM). We evaluated the influence of inherited HAS1 single nucleotide polymorphisms (SNP) on the risk of having a systemic B cell malignancy in 1414 individuals compromising 832 patients and 582 healthy controls, including familial analysis of an Icelandic kindred. We sequenced HAS1 gene segments from 181 patients with MM, 98 with monoclonal gammopathy of undetermined significance (MGUS), 72 with Waldenstrom macroglobulinemia (WM), 169 with chronic lymphocytic leukemia (CLL), as well as 34 members of a monoclonal gammopathy-prone Icelandic family, 212 age-matched healthy donors and a case-control cohort of 295 breast cancer patients with 353 healthy controls. Three linked single nucleotide polymorphisms (SNP) in HAS1 intron3 are significantly associated with B-cell malignancies (range pâ=â0.007 to pâ=â10(-5)), but not MGUS or breast cancer, and predict risk in a 34 member Icelandic family (pâ=â0.005, Odds Ratioâ=â5.8 (OR)), a relatively homogeneous cohort. In contrast, exon3 SNPs were not significantly different among the study groups. Pooled analyses showed a strong association between the linked HAS1 intron3 SNPs and B-cell malignancies (ORâ=â1.78), but not for sporadic MGUS or for breast cancer (OR<1.0). The minor allele genotypes of HAS1 SNPs are significantly more frequent in MM, WM, CLL and in affected members of a monoclonal gammopathy-prone family than they are in breast cancer, sporadic MGUS or healthy donors. These inherited changes may increase the risk for systemic B-cell malignancies but not for solid tumors.