ABSTRACT
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.—Liu, Y., Huang, X., He, X., Zhou, Y., Jiang, X., Chen‐Kiang, S., Jaffrey, S. R., Xu, G. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function. FASEB J. 29, 4829–4839 (2015). www.fasebj.org
BACKGROUND:
Epigenetic dysregulation is a hallmark of cancer, including multiple myeloma. Inhibitors of histone deacetylases (HDACs) induce DNA hyperacetylation by inhibiting removal of acetyl groups ...from amino tails on histone proteins, thereby uncoiling condensed chromatin favoring transcription of silenced genes, including tumor suppressor genes. Romidepsin is an HDAC inhibitor that exhibits antiproliferative and apoptotic effects against multiple myeloma cell lines.
METHODS:
A phase 2 trial was performed of romidepsin in patients with multiple myeloma who were refractory to standard therapy. Treatment was comprised of romidepsin (13 mg/m2) given as a 4‐hour intravenous infusion on Days 1, 8, and 15 every 28 days). Thirteen patients received a median of 2 cycles of therapy (range, 1‐7 cycles).
RESULTS:
Although no patients had an objective response, 4 of 12 patients with secretory myeloma exhibited evidence of M‐protein stabilization, and several other patients experienced improvement in bone pain and resolution of hypercalcemia.
CONCLUSIONS:
The results of the current study demonstrate that romidepsin, as a single agent, is unlikely to be associated with a response rate of ≥30% in patients with refractory myeloma, although there was some clinical evidence suggesting a biological effect associated with therapy. Cancer 2011. Published 2010 by the American Cancer Society.
Romidepsin is an histone deacetylase inhibitor that exhibits antiproliferative and apoptotic effects against multiple myeloma cell lines. A phase 2 trial was performed of romidepsin in patients with multiple myeloma who were refractory to standard therapy. Patients with myeloma exhibited evidence of M‐protein stabilization and improvement in bone pain and resolution of hypercalcemia. It was concluded that romidepsin, as a single agent, had modest activity but may be used in combination with other agents as adjunctive therapy.
Mantle cell lymphoma (MCL) carries an unfavorable prognosis and requires new treatment strategies. The associated t(11:14) translocation results in enhanced cyclin D1 expression and cyclin ...D1–dependent kinase activity to promote cell-cycle progression. A pharmacodynamic study of the selective CDK4/6 inhibitor PD0332991 was conducted in 17 patients with relapsed disease, using 2-deoxy-2-18Ffluoro-D-glucose (FDG) and 3-deoxy-318Ffluorothymidine (FLT) positron emission tomography (PET) to study tumor metabolism and proliferation, respectively, in concert with pre- and on-treatment lymph node biopsies to assess retinoblastoma protein (Rb) phosphorylation and markers of proliferation and apoptosis. Substantial reductions in the summed FLT-PET maximal standard uptake value (SUVmax), as well as in Rb phosphorylation and Ki-67 expression, occurred after 3 weeks in most patients, with significant correlations among these end points. Five patients achieved progression-free survival time of > 1 year (range, 14.9-30.1+ months), with 1 complete and 2 partial responses (18% objective response rate; 90% confidence interval, 5%-40%). These patients demonstrated > 70%, > 90%, and ≥ 87.5% reductions in summed FLT SUVmax and expression of phospho-Rb and Ki67, respectively, parameters necessary but not sufficient for long-term disease control. The results of the present study confirm CDK4/6 inhibition by PD0332991 at a well-tolerated dose and schedule and suggest clinical benefit in a subset of MCL patients. This study is registered at www.clinicaltrials.gov under identifier NCT00420056.
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.
•Ibrutinib 560 mg daily plus palbociclib 100 mg on days 1 to 21 of each 28-day cycle could be safely administered to patients with previously treated MCL.•Complete responses and duration of response (median, >2 years) were high relative to studies of single-agent ibrutinib.
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Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)–induced B-cell transformation to document the ...relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV+ lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.
•EBV infection leads to PRMT5 overexpression and global epigenetic changes that are essential to drive B-lymphocyte transformation.•Highly selective PRMT5 inhibitors represent a novel, first-in-class drug that restores critical regulatory checkpoints in lymphoma cells.
Treatment of mantle-cell lymphoma (MCL) is nonstandardized, though patients are commonly treated immediately at diagnosis. Because data on observation, or "watch and wait," have not been previously ...reported, we analyzed the outcome of deferred initial therapy.
Inclusion criteria in this retrospective analysis were a diagnosis of MCL between 1997 and 2007 and known date of first treatment. Hospital and research charts were reviewed for prognostic and treatment-related information. Date of death was derived from hospital records and confirmed using an online Social Security death index.
Of 97 patients with MCL evaluated at Weill Cornell Medical Center, 31 patients (32%) were observed for more than 3 months before initial systemic therapy, with median time to treatment for the observation group of 12 months (range, 4 to 128 months). The observation group (median follow-up, 55 months) had a median age of 58 years (range, 40 to 81 years). Prognostic factors in assessable patients included advanced stage (III/IV) in 75%, elevated lactate dehydrogenase in 25%, and intermediate- or high-risk Mantle Cell International Prognostic Index in 54%. Better performance status and lower-risk standard International Prognostic Index scores were more commonly present in those undergoing observation. Although time to treatment did not predict overall survival in a multivariate analysis, the survival profile of the observation group was statistically superior to that of the early treatment group (not reached v 64 months, P = .004).
In selected asymptomatic patients with MCL, deferred initial treatment ("watch and wait") is an acceptable management approach.
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.
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/m
2
(dose levels 1, 2, 3) or 1.3 mg/m
2
(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/m
2
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.
Richter's Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine ...methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.
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