ABSTRACT
Proteasome inhibitors and immunomodulatory drugs (IMiDs) are two major types of drugs for the treatment of multiple myeloma. Although different combination therapies for myeloma have been developed and achieved high responsive rate, these strategies frequently result in drug resistance. Therefore, it is necessary to explore new molecular mechanisms and therapeutic approaches to fulfill this unmet medical need. Here, we find that proteasome inhibitor bortezomib (Btz) causes cereblon (CRBN) cleavage and that caspase-8 (CASP-8) is responsible for this cleavage. Either inhibition or genetic depletion of CASP-8 decreased the CRBN cleavage upon Btz treatment, which could potentiate the antimyeloma activity of IMiD lenalidomide (Len). This work suggests that administration of CASP-8 inhibitors might enhance the overall effectiveness of Btz/Len-based therapeutic treatment of patients with myeloma. SIGNIFICANCE STATEMENT: Caspase-8 activation upon bortezomib treatment results in the cleavage of cereblon, a substrate receptor of the cullin-4 RING E3 ligase, which is responsible for the degradation of two transcription factors, Ikaros family zinc finger protein (IKZF) 1 and IKZF3, in the presence of immunomodulatory drugs including lenalidomide. The administration of caspase-8 inhibitor may enhance the antimyeloma activity of the combination therapy with bortezomib and lenalidomide.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Antineoplastic Agents/administration & dosage , Bortezomib/administration & dosage , Caspase 8/metabolism , Lenalidomide/administration & dosage , Multiple Myeloma/enzymology , Caspase 8/genetics , Caspase Inhibitors/pharmacology , Drug Therapy, Combination , HEK293 Cells , Humans , K562 Cells , Multiple Myeloma/geneticsABSTRACT
Single-agent ibrutinib is active in patients with previously treated mantle cell lymphoma (MCL); however, nearly half of all patients experience treatment failure during the first year. We previously demonstrated that prolonged early G1 cell cycle arrest induced by the oral, specific CDK4/6 inhibitor palbociclib can overcome ibrutinib resistance in primary human MCL cells and MCL cell lines expressing wild-type Bruton's tyrosine kinase (BTK). Therefore, we conducted a phase 1 trial to evaluate the dosing, safety, and preliminary activity of palbociclib plus ibrutinib in patients with previously treated mantle cell lymphoma. From August 2014 to June 2016, a total of 27 patients (21 men, 6 women) were enrolled. The maximum tolerated doses were ibrutinib 560 mg daily plus palbociclib 100 mg on days 1 to 21 of each 28-day cycle. The dose-limiting toxicity was grade 3 rash. The most common grade 3 to 4 toxicities included neutropenia (41%), thrombocytopenia (30%), hypertension (15%), febrile neutropenia (15%), and lung infection (11%). The overall and complete response rates were 67% and 37%, and with a median follow-up of 25.6 months, the 2-year progression-free survival was 59.4% and the 2-year response duration was 69.8%. A phase 2 multicenter clinical trial to further characterize efficacy is now ongoing. The current trial was registered at www.clinicaltrials.gov as #NCT02159755.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Lymphoma, Mantle-Cell/drug therapy , Neoplasm Recurrence, Local/drug therapy , Adenine/analogs & derivatives , Adult , Aged , Aged, 80 and over , Female , Follow-Up Studies , Humans , Lymphoma, Mantle-Cell/pathology , Male , Maximum Tolerated Dose , Middle Aged , Neoplasm Recurrence, Local/pathology , Piperazines/administration & dosage , Piperidines , Prognosis , Pyrazoles/administration & dosage , Pyridines/administration & dosage , Pyrimidines/administration & dosage , Survival RateABSTRACT
The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 µM) and its structural analog lenalidomide (10 µM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.
Subject(s)
Multiple Myeloma/metabolism , Peptide Hydrolases/metabolism , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Adaptor Proteins, Signal Transducing , Cell Line, Tumor , HEK293 Cells , Humans , Lenalidomide , Multiple Myeloma/pathology , Peptide Hydrolases/geneticsABSTRACT
Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.
Subject(s)
Apoptosis/drug effects , G1 Phase Cell Cycle Checkpoints/drug effects , Interferon Regulatory Factors/genetics , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/pharmacology , Animals , Apoptosis/genetics , Boronic Acids/administration & dosage , Boronic Acids/pharmacology , Bortezomib , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cytotoxins/administration & dosage , Cytotoxins/pharmacology , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Resistance, Neoplasm/drug effects , Drug Synergism , G1 Phase Cell Cycle Checkpoints/genetics , G1 Phase Cell Cycle Checkpoints/physiology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Interferon Regulatory Factors/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Protein Kinase Inhibitors/administration & dosage , Pyrazines/administration & dosage , Pyrazines/pharmacology , Substrate Specificity , Time Factors , Xenograft Model Antitumor AssaysABSTRACT
Inhibition of Cdk4/Cdk6 by p18(INK4c) (p18) is pivotal for generation of noncycling immunoglobulin (Ig)-secreting plasma cells (PCs). In the absence of p18, CD138(+) plasmacytoid cells continue to cycle and turnover rapidly, suggesting that p18 controls PC homeostasis. We now show that p18 selectively acts in a rare population of rapidly cycling CD138(hi)/B220(hi) intermediate PCs (iPCs). While retaining certain B-cell signatures, iPCs are poised to differentiate to end-stage PCs although the majority undergo apoptosis. p18 is dispensable for the development of the PC transcriptional circuitry, and Blimp-1 and Bcl-6 are expressed fully and mutually exclusively in individual iPCs. However, a minor proportion of iPCs express both, and they are preferentially protected by p18 or Bcl-xL overexpression, consistent with expansion of the iPC pool by Bcl-xL overexpression, or loss of proapoptotic Bim or Noxa. Expression of Noxa is induced during B-cell activation, peaks in iPCs, and selectively repressed by p18. It is required to promote apoptosis of cycling B cells, especially in the absence of p18. These findings define the first physiologic function for Noxa and suggest that by repressing Noxa, induction of G1 arrest by p18 bypasses a homeostatic cell-cycle checkpoint in iPCs for PC differentiation.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/physiology , Cyclin-Dependent Kinase Inhibitor p18/physiology , Plasma Cells/cytology , Plasma Cells/physiology , Proto-Oncogene Proteins c-bcl-2/physiology , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Cycle/genetics , Cell Cycle/physiology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cyclin-Dependent Kinase Inhibitor p18/deficiency , Cyclin-Dependent Kinase Inhibitor p18/genetics , HEK293 Cells , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Homeostasis/genetics , Homeostasis/physiology , Humans , Immunoglobulin G/biosynthesis , Leukocyte Common Antigens/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Models, Biological , Positive Regulatory Domain I-Binding Factor 1 , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/deficiency , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-6/genetics , Proto-Oncogene Proteins c-bcl-6/physiology , RNA, Small Interfering/genetics , Syndecan-1/metabolism , Transcription Factors/genetics , Transcription Factors/physiology , bcl-X Protein/genetics , bcl-X Protein/physiologyABSTRACT
Telomere length maintenance is essential for cellular immortalization and tumorigenesis. 5% - 10% of human cancers rely on a recombination-based mechanism termed alternative lengthening of telomeres (ALT) to sustain their replicative immortality, yet there are currently no targeted therapies. Through CRISPR/Cas9-based genetic screens in an ALT-immortalized isogenic cellular model, here we identify histone lysine demethylase KDM2A as a molecular vulnerability selectively for cells contingent on ALT-dependent telomere maintenance. Mechanistically, we demonstrate that KDM2A is required for dissolution of the ALT-specific telomere clusters following homology-directed telomere DNA synthesis. We show that KDM2A promotes de-clustering of ALT multitelomeres through facilitating isopeptidase SENP6-mediated SUMO deconjugation at telomeres. Inactivation of KDM2A or SENP6 impairs post-recombination telomere de-SUMOylation and thus dissolution of ALT telomere clusters, leading to gross chromosome missegregation and mitotic cell death. These findings together establish KDM2A as a selective molecular vulnerability and a promising drug target for ALT-dependent cancers.
ABSTRACT
Telomere length maintenance is essential for cellular immortalization and tumorigenesis. 5% - 10% of human cancers rely on a recombination-based mechanism termed alternative lengthening of telomeres (ALT) to sustain their replicative immortality, yet there are currently no targeted therapies. Through CRISPR/Cas9-based genetic screens in an ALT-immortalized isogenic cellular model, here we identify histone lysine demethylase KDM2A as a molecular vulnerability selectively for cells contingent on ALT-dependent telomere maintenance. Mechanistically, we demonstrate that KDM2A is required for dissolution of the ALT-specific telomere clusters following recombination-directed telomere DNA synthesis. We show that KDM2A promotes de-clustering of ALT multitelomeres through facilitating isopeptidase SENP6-mediated SUMO deconjugation at telomeres. Inactivation of KDM2A or SENP6 impairs post-recombination telomere de-SUMOylation and thus dissolution of ALT telomere clusters, leading to gross chromosome missegregation and mitotic cell death. These findings together establish KDM2A as a selective molecular vulnerability and a promising drug target for ALT-dependent cancers.
Subject(s)
F-Box Proteins , Neoplasms , Telomerase , Humans , Cell Line , DNA , Telomere Homeostasis/genetics , Telomere/genetics , Telomere/metabolism , Neoplasms/genetics , Telomerase/genetics , Cysteine Endopeptidases/metabolism , F-Box Proteins/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolismABSTRACT
Targeting lineage-defined transcriptional dependencies has emerged as an effective therapeutic strategy in cancer treatment. Through screening for molecular vulnerabilities of mantle cell lymphoma (MCL), we identified a set of transcription factors (TFs) including FOXO1, EBF1, PAX5, and IRF4 that are essential for MCL propagation. Integrated chromatin immunoprecipitation and sequencing (ChIP-Seq) with transcriptional network reconstruction analysis revealed FOXO1 as a master regulator that acts upstream in the regulatory TF hierarchy. FOXO1 is both necessary and sufficient to drive MCL lineage commitment through supporting the lineage-specific transcription programs. We further show that FOXO1, but not its close paralog FOXO3, can reprogram myeloid leukemia cells and induce B-lineage gene expression. Finally, we demonstrate that cpd10, a small molecule identified from an enriched FOXO1 inhibitor library, induces a robust cytotoxic response in MCL cells in vitro and suppresses MCL progression in vivo. Our findings establish FOXO1 inhibition as a therapeutic strategy targeting lineage-driven transcriptional addiction in MCL.
Subject(s)
Lymphoma, Mantle-Cell , Humans , Adult , Lymphoma, Mantle-Cell/genetics , Gene Regulatory Networks , Forkhead Box Protein O1/geneticsABSTRACT
Cell cycle dysregulation caused by aberrant cyclin D1 and CDK4 expression is a major determinant for proliferation of cancer cells in mantle cell lymphoma (MCL). Inhibition of CDK4/6 induces G1 arrest of MCL cells in patients, appearing to deepen and prolong the clinical response to partner agents. This article reviews aberrations of cell cycle genes in MCL cells and clinical trials of CDK4/6 inhibitors for MCL. Integrative longitudinal functional genomics is discussed as a strategy to discover genomic drivers for resistance in cancer cells and cancer-immune interactions that potentially contribute to the clinical response to palbociclib combination therapy in MCL.
Subject(s)
Cyclin D1 , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , G1 Phase Cell Cycle Checkpoints , Lymphoma, Mantle-Cell , Piperazines/therapeutic use , Pyridines/therapeutic use , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , G1 Phase Cell Cycle Checkpoints/drug effects , G1 Phase Cell Cycle Checkpoints/genetics , Genomics , Humans , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/pathologyABSTRACT
Targeting the cell cycle represents a rational approach to mantle cell lymphoma (MCL) therapy, as aberrant expression of cyclin D1 and dysregulation of CDK4 underlie cell cycle progression and proliferation of MCL cells. Although cell cycle cancer therapy was historically ineffective due to a lack of selective and effective drugs, this landscape changed with the advent of selective and potent small-molecule oral CDK4/6 inhibitors. Here, we review the anti-tumor activities and clinical data of selective CDK4/6 inhibitors in MCL. We summarize the known mechanism of action of palbociclib, the most specific CDK4/6 inhibitor to date, and the strategy to leverage this specificity to reprogram MCL for a deeper and more durable clinical response to partner drugs. We also discuss integrative longitudinal functional genomics as a strategy to discover tumor-intrinsic genomic biomarkers and tumor-immune interactions that potentially contribute to the clinical response to palbociclib in combination therapy for MCL. Understanding the genomic basis for targeting CDK4/6 and the mechanisms of action and resistance in MCL may advance personalized therapy for MCL and shed light on drug resistance in other cancers.
ABSTRACT
Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4), mediates the ubiquitination and degradation of constitutive substrates and immunomodulatory drug-induced neo-substrates including MEIS2, c-Jun, CLC1, IKZF1/3, CK1α, and SALL4. It has been reported that CRBN itself could be degraded through the ubiquitin-proteasome system by its associated or other cullin-RING E3 ligases, thus influencing its biological functions. However, it is unknown whether the CRBN stability and its biological function could be modulated by caspases. In this study, using model cell lines, we found that activation of the death receptor using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) leads to the decreased CRBN protein level. Through pharmacological inhibition and activation of caspase-8 (CASP-8), we disclosed that CASP-8 regulates CRBN cleavage in cell lines. Site mapping experiments revealed that CRBN is cleaved after Asp9 upon CASP-8 activation, resulting in the reduced stability. Using myeloma as a model system, we further revealed that either inhibition or genetic depletion of CASP-8 enhances the anti-myeloma activity of lenalidomide (Len) by impairing CRBN cleavage, leading to the attenuated IKZF1 and IKZF3 protein levels and the reduced viability of myeloma cell lines and primary myeloma cells from patients. The present study discovered that the stability of the substrate receptor of an E3 ligase can be modulated by CASP-8 and suggested that administration of CASP-8 inhibitors enhances the overall effectiveness of Len-based combination therapy in myeloma.
ABSTRACT
In mantle cell lymphoma (MCL), cyclin D1 combines with CDK4/6 to phosphorylate Rb, releasing a break on the G1 to S phase cell cycle. Palbociclib is a specific, potent, oral inhibitor of CDK4/6 capable of inducing a complete, prolonged G1 cell cycle arrest (pG1) in Rb+ MCL cells. The proteasome inhibitor bortezomib is approved by the US Food and Drug Administration for treatment of mantle cell lymphoma. Palbociclib-induced pG1 appears to sensitize MCL cells to killing by low-dose bortezomib, potentially improving its activity and tolerability. We conducted a phase 1 trial of palbociclib plus bortezomib in patients with previously treated MCL (NCT01111188). Patients received palbociclib at 75 mg (dose level 1), 100 mg (dose level 2), or 125 mg (dose levels 3 and 4) on days 1-12 of each 21-day cycle in addition to intravenous bortezomib 1.0 mg/m2 (dose levels 1, 2, 3) or 1.3 mg/m2 (dose level 4) on days 8, 11, 15 and 18. A total of 19 patients with a median age of 64 and an average of 2 prior therapies were enrolled. Two subjects experienced dose limiting toxicity (DLT): thrombocytopenia (dose level 1) and neutropenia (dose level 3). Although no DLTs were seen at dose level 4, all patients required dose delays during cycle 2 due to cytopenias, and the study team decided to stop the trial. Four of 19 patients achieved a clinical response, including one patient with a complete response. Three patients received treatment for more than one year, including one patient receiving single-agent palbociclib for more than 6 years. The combination of palbociclib 125 mg on days 1-12 plus bortezomib 1.0 mg/m2 on days 8, 11, 15, and 18 of a 21-day cycle is feasible and active in previously treated MCL, with the primary toxicity being myelosuppression. The regimen may be worthy of further evaluation in patients with non-blastoid MCL following failure of other newer agents.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Lymphoma, Mantle-Cell/drug therapy , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Bortezomib/administration & dosage , Bortezomib/pharmacokinetics , Drug Administration Schedule , Drug Monitoring , Female , Humans , Lymphoma, Mantle-Cell/diagnosis , Male , Middle Aged , Piperazines/administration & dosage , Piperazines/pharmacokinetics , Pyridines/administration & dosage , Pyridines/pharmacokinetics , Retreatment , Treatment OutcomeABSTRACT
Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S-phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C). This sensitization is achieved in part through enrichment of S-phase cells, which maximizes the AML populations for Ara-C incorporation into replicating DNA to elicit DNA damage. Moreover, PD 0332991 triggered apoptosis of AML cells through inhibition of the homeobox (HOX)A9 oncogene expression, reducing the transcription of its target PIM1. Reduced PIM1 synthesis attenuates PIM1-mediated phosphorylation of the proapoptotic BAD and activates BAD-dependent apoptosis. In vivo, timely inhibition of CDK4/CDK6 by PD 0332991 and release profoundly suppresses tumor growth in response to reduced doses of Ara-C in a xenograft AML model. Collectively, these data suggest selective and reversible inhibition of CDK4/CDK6 as an effective means to enhance Ara-C killing of AML cells at reduced doses, which has implications for the treatment of elderly AML patients who are unable to tolerate high-dose Ara-C therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Line , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Cytarabine/pharmacology , DNA Damage/drug effects , DNA Replication/drug effects , HEK293 Cells , HL-60 Cells , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice, Inbred NOD , Mice, SCID , Phosphorylation/drug effects , Proto-Oncogene Proteins c-pim-1/genetics , Proto-Oncogene Proteins c-pim-1/metabolism , S Phase/drug effects , S Phase/genetics , Transcription, Genetic/drug effects , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
This phase 1/2 study was the first to evaluate the safety and efficacy of the cyclin-dependent kinase (CDK) 4/6-specific inhibitor palbociclib (PD-0332991) in sequential combination with bortezomib and dexamethasone in relapsed/refractory multiple myeloma. The recommended phase 2 dose was palbociclib 100 mg orally once daily on days 1-12 of a 21-day cycle with bortezomib 1.0 mg/m2 (intravenous) and dexamethasone 20 mg (orally 30 min pre-bortezomib dosing) on days 8 and 11 (early G1 arrest) and days 15 and 18 (cell cycle resumed). Dose-limiting toxicities were primarily cytopenias; most other treatment-related adverse events were grade≤3. At a bortezomib dose lower than that in other combination therapy studies, antitumor activity was observed (phase 1). In phase 2, objective responses were achieved in 5 (20%) patients; 11 (44%) achieved stable disease. Biomarker and pharmacodynamic assessments demonstrated that palbociclib inhibited CDK4/6 and the cell cycle initially in most patients.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Bortezomib , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Dexamethasone/administration & dosage , Drug Administration Schedule , Drug Monitoring , Drug Resistance, Neoplasm , Female , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Multiple Myeloma/metabolism , Multiple Myeloma/mortality , Neoplasm Staging , Piperazines/administration & dosage , Pyridines/administration & dosage , Recurrence , Retreatment , Treatment OutcomeABSTRACT
UNLABELLED: Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. SIGNIFICANCE: We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973.
Subject(s)
Cell Cycle/genetics , Genomics , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Mutation , Phosphoinositide-3 Kinase Inhibitors , Protein-Tyrosine Kinases/genetics , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase , Amino Acid Substitution , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , DNA Mutational Analysis , Drug Resistance, Neoplasm/genetics , Drug Synergism , Enzyme Activation , Humans , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/pathology , NF-kappa B/metabolism , Neoplasm Recurrence, Local , Nitrates/pharmacology , Nitrates/therapeutic use , Piperidines , Polyethylene Glycols/pharmacology , Polyethylene Glycols/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcr/metabolism , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Signal Transduction , Treatment OutcomeABSTRACT
Phosphatidylinositol-3-kinase (PI3K) signaling is constitutive in most human cancers. Selective inhibition of PI3Kδ (p110δ) by GS-1101 has emerged as a promising therapy in chronic lymphocytic leukemia and indolent lymphomas. In aggressive non-Hodgkin lymphomas such as mantle cell lymphoma (MCL), however, efficacy has been observed, but the extent and duration of tumor control is modest. To determine if tumor killing by GS-1101 is cell cycle-dependent, we show in primary MCL cells by whole-transcriptome sequencing that, despite aberrant expression and recurrent mutations in Cyclin D1, mutations are rare in coding regions of CDK4, RB1 and other genes that control G1-S cell cycle progression or PI3K/AKT signaling. PI3Kδ is the predominant PI3K catalytic subunit expressed, and inhibition by GS-1101 transiently inhibits AKT phosphorylation but not proliferation in MCL cells. Induction of prolonged early G1-arrest (pG1) by selective inhibition of CDK4/CDK6 with PD 0332991 amplifies and sustains PI3Kδ inhibition, which leads to robust apoptosis. Accordingly, inhibition of PI3Kδ induces apoptosis of primary MCL tumor cells once they have ceased to cycle ex vivo, and this killing is enhanced by PD 0332991 inhibition of CDK4/CDK6. PIK3IP1, a negative PI3K regulator, appears to mediate pG1 sensitization to PI3K inhibition; it is markedly reduced in MCL tumor cells compared with normal peripheral B cells, profoundly induced in pG1 and required for pG1 sensitization to GS-1101. Thus, the magnitude and duration of PI3K inhibition and tumor killing by GS-1101 is pG1-dependent, suggesting induction of pG1 by CDK4/CDK6 inhibition as a strategy to sensitize proliferating lymphoma cells to PI3K inhibition.
Subject(s)
Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , G1 Phase Cell Cycle Checkpoints/drug effects , Lymphoma/metabolism , Membrane Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , G1 Phase Cell Cycle Checkpoints/genetics , Humans , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Mutation/genetics , Phosphatidylinositol 3-Kinases/genetics , Piperazines/pharmacology , Purines/pharmacology , Pyridines/pharmacology , Quinazolinones/pharmacologyABSTRACT
PURPOSE: The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67(+) malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. EXPERIMENTAL DESIGN: The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. RESULTS: MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms. CONCLUSIONS: These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms.
Subject(s)
Antigens, Neoplasm/metabolism , Apoptosis , Gene Expression Regulation, Neoplastic , Inhibitor of Apoptosis Proteins/metabolism , Multiple Myeloma/metabolism , Neoplasm Proteins/metabolism , bcl-2-Associated X Protein/metabolism , Antigens, Neoplasm/genetics , Apoptosis/genetics , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Disease Progression , Female , Gene Silencing , Humans , Inhibitor of Apoptosis Proteins/genetics , Male , Middle Aged , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Proteins/genetics , Signal Transduction/genetics , Survivin , Tumor Suppressor Protein p53/metabolismABSTRACT
Multiple myeloma (MM) remains incurable partly because no effective cell cycle-based therapy has been available to both control tumor cell proliferation and synergize with cytotoxic killing. PD 0332991 is an orally active small molecule that potently and specifically inhibits Cdk4 and Cdk6. It has been shown to induce rapid G(1) cell cycle arrest in primary human myeloma cells and suppress tumor growth in xenograft models. To improve therapeutic targeting of myeloma progression, we combined tumor suppression by PD 0332991 with cytotoxic killing by bortezomib, a proteasome inhibitor widely used in myeloma treatment, in the immunocompetent 5T33MM myeloma model. We show that 5T33MM tumor cells proliferate aggressively in vivo due to expression of cyclin D2, elevation of Cdk4, and impaired p27(Kip1) expression, despite inhibition of Cdk4/6 by p18(INK4c) and the maintenance of a normal plasma cell transcription program. PD 0332991 potently inhibits Cdk4/6-specific phosphorylation of Rb and cell cycle progression through G(1) in aggressively proliferating primary 5T33MM cells, in vivo and ex vivo. This leads to tumor suppression and a significant improvement in survival. Moreover, induction of G(1) arrest by PD 0332991 sensitizes 5T33MM tumor cells to killing by bortezomib. Inhibition of Cdk4/6 by PD 0332991, therefore, effectively controls myeloma tumor expansion and sensitizes tumor cells to bortezomib killing in the presence of an intact immune system, thereby representing a novel and promising cell cycle-based combination therapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Boronic Acids/administration & dosage , Multiple Myeloma/drug therapy , Piperazines/administration & dosage , Pyrazines/administration & dosage , Pyridines/administration & dosage , Animals , Apoptosis , Bortezomib , Cell Cycle , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Enzyme Inhibitors/administration & dosage , Humans , Mice , Mice, Inbred C57BL , Neoplasm TransplantationABSTRACT
Cell-cycle entry is critical for homeostatic control in physiologic response of higher organisms but is not well understood. The antibody response begins with induction of naive mature B cells, which are naturally arrested in G(0)/G(1) phase of the cell cycle, to enter the cell cycle in response to antigen and cytokine. BLyS (BAFF), a cytokine essential for mature B cell development and survival, is thought to act mainly by attenuation of apoptosis. Here, we show that BLyS alone induces cell-cycle entry and early G(1) cell-cycle progression, but not S-phase entry, in opposition to the cyclin-dependent kinase inhibitors p18(INK4c). Independent of its survival function, BLyS enhances the synthesis of cyclin D2, in part through activation of NF-kappaB, as well as CDK4 and retinoblastoma protein phosphorylation. By convergent activation of the same cell-cycle regulators in opposition to p18(INK4c), B cell receptor signaling induces cell-cycle entry and G(1) progression in synergy with BLyS, but also DNA replication. The failure of BLyS to induce S-phase cell-cycle entry lies in its inability to increase cyclin E and reduce p27(Kip1) expression. Antagonistic cell-cycle regulation by BLyS and p18(INK4c) is functionally linked to apoptotic control and conserved from B cell activation in vitro to antibody response in vivo, further indicating a physiologic role in homeostasis.