Besides many other mutations in known cancer driver genes, mantle cell lymphoma (MCL) is characterized by recurrent genetic alterations of important regulators of the phosphoinositol-3-kinase (PI3K) cascade including PIK3CA gains and PTEN losses. To evaluate the biological and functional consequences of these aberrations in MCL, we have introduced transgenic expression of PIK3CA (PIK3CA UP) and performed knockout/knockdown of PTEN gene (PTEN KO/KD) in 5 MCL cell lines. The modified cell lines were tested for associated phenotypes including dependence on upstream B-cell receptor (BCR) signaling (by an additional BCR knockout). PIK3CA overexpression decreased the dependence of the tested MCL on prosurvival signaling from BCR, decreased levels of oxidative phosphorylation, and increased resistance to 2-deoxy-glucose, a glycolysis inhibitor. Unchanged protein kinase B (AKT) phosphorylation status and unchanged sensitivity to a battery of PI3K inhibitors suggested that PIK3CA gain might affect MCL cells in AKT-independent manner. PTEN KO was associated with a more distinct phenotype: AKT hyperphosphorylation and overactivation, increased resistance to multiple inhibitors (most of the tested PI3K inhibitors, Bruton tyrosine kinase inhibitor ibrutinib, and BCL2 inhibitor venetoclax), increased glycolytic rates with resistance to 2-deoxy-glucose, and significantly decreased dependence on prosurvival BCR signaling. Our results suggest that the frequent aberrations of the PI3K pathway may rewire associated signaling with lower dependence on BCR signaling, better metabolic and hypoxic adaptation, and targeted therapy resistance in MCL.
- MeSH
- chemorezistence genetika MeSH
- cílená molekulární terapie MeSH
- fosfatidylinositol-3-kinasy třídy I * genetika metabolismus MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- fosfohydroláza PTEN * metabolismus genetika MeSH
- lidé MeSH
- lymfom z plášťových buněk * farmakoterapie genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- receptory antigenů B-buněk metabolismus MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Venetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which call for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient-derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL2L11/BIM status.
- MeSH
- antitumorózní látky farmakologie terapeutické užití MeSH
- apoptóza účinky léků MeSH
- benzothiazoly MeSH
- bicyklické sloučeniny heterocyklické * farmakologie terapeutické užití MeSH
- chemorezistence * MeSH
- isochinoliny MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- protein bcl-X * metabolismus antagonisté a inhibitory MeSH
- protein BCL2L11 * metabolismus genetika MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus antagonisté a inhibitory MeSH
- sulfonamidy * farmakologie terapeutické užití MeSH
- synergismus léků MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Mantle cell lymphoma (MCL) is a chronically relapsing malignancy with deregulated cell cycle progression. We analyzed efficacy, mode of action, and predictive markers of susceptibility to palbociclib, an approved CDK 4/6 inhibitor, and its combination with venetoclax, a BCL2 inhibitor. METHODS: A panel of nine MCL cell lines were used for in vitro experiments. Four patient derived xenografts (PDX) obtained from patients with chemotherapy and ibrutinib-refractory MCL were used for in vivo proof-of-concept studies. Changes of the mitochondrial membrane potential, energy-metabolic pathways, AKT activity, and pro-apoptotic priming of MCL cells were evaluated by JC-1 staining, Seahorse XF analyser, genetically encoded fluorescent AKT reporter, and BH3 profiling, respectively. MCL clones with gene knockout or transgenic (over)expression of CDKN2A, MYC, CDK4, and RB1 were used to estimate impact of these aberrations on sensitivity to palbociclib, and venetoclax. RESULTS: Co-targeting MCL cells with palbociclib and venetoclax induced cytotoxic synergy in vitro and in vivo. Molecular mechanisms responsible for the observed synthetic lethality comprised palbociclib-mediated downregulation of anti-apoptotic MCL1, increased levels of proapoptotic BIM bound on both BCL2, and BCL-XL and increased pro-apoptotic priming of MCL cells mediated by BCL2-independent mechanisms, predominantly palbociclib-triggered metabolic and mitochondrial stress. Loss of RB1 resulted in palbociclib resistance, while deletion of CDKN2A or overexpression of CDK4, and MYC genes did not change sensitivity to palbociclib. CONCLUSIONS: Our data strongly support investigation of the chemotherapy-free palbociclib and venetoclax combination as an innovative treatment strategy for post-ibrutinib MCL patients without RB1 deletion.
Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin lymphoma, is characterized by an aggressive clinical course. In approximately one-third of patients with DLBCL, first-line multiagent immunochemotherapy fails to produce a durable response. Molecular heterogeneity and apoptosis resistance pose major therapeutic challenges in DLBCL treatment. To circumvent apoptosis resistance, the induction of ferroptosis might represent a promising strategy for lymphoma therapy. In this study, a compound library, targeting epigenetic modulators, was screened to identify ferroptosis-sensitizing drugs. Strikingly, bromodomain and extra-terminal domain (BET) inhibitors sensitized cells of the germinal center B-cell-like (GCB) subtype of DLBCL to ferroptosis induction and the combination of BET inhibitors with ferroptosis inducers, such as dimethyl fumarate or RSL3, synergized in the killing of DLBCL cells in vitro and in vivo. On the molecular level, the BET protein BRD4 was found to be an essential regulator of ferroptosis suppressor protein 1 expression and thus to protect GCB-DLBCL cells from ferroptosis. Collectively, we identified and characterized BRD4 as an important player in ferroptosis suppression in GCB-DLBCL and provide a rationale for the combination of BET inhibitors with ferroptosis-inducing agents as a novel therapeutic approach for DLBCL treatment.
FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.
- MeSH
- akutní myeloidní leukemie * patologie MeSH
- antitumorózní látky * farmakologie terapeutické užití MeSH
- apoptóza MeSH
- inhibitory proteinkinas farmakologie terapeutické užití MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- pyridaziny * farmakologie terapeutické užití MeSH
- pyrimidiny farmakologie MeSH
- tyrosinkinasa 3 podobná fms genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Therapy of FLT3-positive acute myeloid leukemia still remains complicated, despite the availability of newly approved kinase inhibitors. Various strategies to avoid the reduced efficacy of therapy have been explored, including the development of dual targeting compounds, which inhibit FLT3 and another kinase necessary for the survival and proliferation of AML cells. We have designed new 2,7,9-trisubstituted 8-oxopurines as FLT3 inhibitors and report here the structure-activity relationship studies. We demonstrated that substituents at positions 7 and 9 modulate activity between CDK4 and FLT3 kinase, and the isopropyl group at position 7 substantially increased the selectivity toward FLT3 kinase, which led to the discovery of compound 15a (9-cyclopentyl-7-isopropyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one). Cellular analyses in MV4-11 cells revealed inhibition of autophosphorylation of FLT3 kinase in nanomolar doses, including the suppression of downstream STAT5 and ERK1/2 phosphorylation. We also describe mechanistic studies in cell lines and activity in a mouse xenograft model in vivo.
- MeSH
- akutní myeloidní leukemie * farmakoterapie MeSH
- apoptóza MeSH
- fosforylace MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- proliferace buněk MeSH
- tyrosinkinasa 3 podobná fms MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cancer therapy failure is a fundamental challenge in cancer treatment. One of the most common reasons for therapy failure is the development of acquired resistance of cancer cells. DNA-damaging agents are frequently used in first-line chemotherapy regimens and DNA damage response, and DNA repair pathways are significantly involved in the mechanisms of chemoresistance. MRE11, a part of the MRN complex involved in double-strand break (DSB) repair, is connected to colorectal cancer (CRC) patients' prognosis. Our previous results showed that single-nucleotide polymorphisms (SNPs) in the 3' untranslated region (3'UTR) microRNA (miRNA) binding sites of MRE11 gene are associated with decreased cancer risk but with shorter survival of CRC patients, which implies the role of miRNA regulation in CRC. The therapy of colorectal cancer utilizes oxaliplatin (oxalato(trans-l-1,2-diaminocyclohexane)platinum), which is often compromised by chemoresistance development. There is, therefore, a crucial clinical need to understand the cellular processes associated with drug resistance and improve treatment responses by applying efficient combination therapies. The main aim of this study was to investigate the effect of miRNAs on the oxaliplatin therapy response of CRC patients. By the in silico analysis, miR-140 was predicted to target MRE11 and modulate CRC prognosis. The lower expression of miR-140 was associated with the metastatic phenotype (p < 0.05) and poor progression-free survival (odds ratio (OR) = 0.4, p < 0.05). In the in vitro analysis, we used miRNA mimics to increase the level of miR-140 in the CRC cell line. This resulted in decreased proliferation of CRC cells (p < 0.05). Increased levels of miR-140 also led to increased sensitivity of cancer cells to oxaliplatin (p < 0.05) and to the accumulation of DNA damage. Our results, both in vitro and in vivo, suggest that miR-140 may act as a tumor suppressor and plays an important role in DSB DNA repair and, consequently, CRC therapy response.
- Publikační typ
- časopisecké články MeSH
Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDXs) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDXs keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We carried out a comparative genetic and histopathological study of 15 PDX models derived from patients with various types of NHL including diffuse large B-cell lymphoma (DLBCL; n = 7), Burkitt lymphoma (BL; n = 1), mantle cell lymphoma (MCL; n = 2), and peripheral T-cell lymphomas (PTCL; n = 5). Whole exome sequencing (WES) of the PDXs and primary lymphoma cells was implemented in 13 out of 15 cases with available DNA samples. Standard immunohistochemistry (IHC) was used to analyze the composition of PDX TME. WES data confirmed that PDXs maintained the genetic heterogeneity with the original primary lymphoma cells. In contrast, IHC analysis revealed the following recurrently observed alterations in the composition of PDX tumors: more blastoid lymphoma cell morphology, increased proliferation rate, lack of non-malignant cellular components including T cells and (human or murine) macrophages, and significantly lower intratumoral microvessel density and microvessel area composed of murine vessels. In addition, PDX tumors derived from T-NHL displayed additional differences compared to the primary lymphoma samples including markedly lower desmoplasia (i.e., the extent of both reticular and collagen fibrosis), loss of expression of cytotoxic granules (i.e., perforin, TIA, granzyme B), or loss of expression of T-cell specific antigens (i.e., CD3, CD4, CD8). Our data suggest that despite keeping the same genetic profiles, PDX models of aggressive NHL do not recapitulate the microenvironmental heterogeneity of the original lymphomas. These findings have implications on the relevance of PDX models in the context of preclinical research.
- MeSH
- antitumorózní látky * MeSH
- difúzní velkobuněčný B-lymfom * MeSH
- dospělí MeSH
- heterografty MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nádorové mikroprostředí MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDXs) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDXs keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We carried out a comparative genetic and histopathological study of 15 PDX models derived from patients with various types of NHL including diffuse large B-cell lymphoma (DLBCL; n = 7), Burkitt lymphoma (BL; n = 1), mantle cell lymphoma (MCL; n = 2), and peripheral T-cell lymphomas (PTCL; n = 5). Whole exome sequencing (WES) of the PDXs and primary lymphoma cells was implemented in 13 out of 15 cases with available DNA samples. Standard immunohistochemistry (IHC) was used to analyze the composition of PDX TME. WES data confirmed that PDXs maintained the genetic heterogeneity with the original primary lymphoma cells. In contrast, IHC analysis revealed the following recurrently observed alterations in the composition of PDX tumors: more blastoid lymphoma cell morphology, increased proliferation rate, lack of non-malignant cellular components including T cells and (human or murine) macrophages, and significantly lower intratumoral microvessel density and microvessel area composed of murine vessels. In addition, PDX tumors derived from T-NHL displayed additional differences compared to the primary lymphoma samples including markedly lower desmoplasia (i.e., the extent of both reticular and collagen fibrosis), loss of expression of cytotoxic granules (i.e., perforin, TIA, granzyme B), or loss of expression of T-cell specific antigens (i.e., CD3, CD4, CD8). Our data suggest that despite keeping the same genetic profiles, PDX models of aggressive NHL do not recapitulate the microenvironmental heterogeneity of the original lymphomas. These findings have implications on the relevance of PDX models in the context of preclinical research.
- MeSH
- antitumorózní látky * MeSH
- difúzní velkobuněčný B-lymfom * genetika MeSH
- dospělí MeSH
- imunohistochemie MeSH
- lidé MeSH
- myši MeSH
- nádorové mikroprostředí genetika MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
