Rational combination treatment with histone deacetylase inhibitors and immunomodulatory drugs in multiple myeloma.
ABSTRACT: Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.
Project description:Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. We have shown that CRBN is also targeted for degradation by SCFFbxo7 ubiquitin ligase. In the current study, we explored the mechanisms underlying sensitivity of MM cells to IMiDs using genome-wide CRISPR-Cas9 screening. We validate that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, inhibits SCFFbxo7 E3 ligase-mediated CRBN degradation, thereby conferring sensitivity to IMiDs; conversely, loss of function of CSN9 signalosome activates SCFFbxo7 complex, thereby enhancing degradation of CRBN and conferring IMiD resistance. Finally, we show that pretreatment with either proteasome inhibitors or NEDD8 activating enzyme (NAE) inhibitors can abrogate degradation and maintain levels of CRBN, thereby enhancing sensitivity to IMiDs. These studies therefore demonstrate that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression.
Project description:Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma. Overall design: Gene expression analysis by RNAseq in multiple myeloma cells OPM-1 treated with DMSO, lenalidomide (LEN), AI-10-104 (RUNXi) or combination.
Project description:Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation, and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.
Project description:Clinical interest in the measurement of Cereblon (CRBN), the primary target of the IMiDs immunomodulatory drugs lenalidomide and pomalidomide, has been fueled by its essential requirement for antitumor or immunomodulatory activity of both drugs in multiple myeloma (MM). However, limited analyses of clinical samples for CRBN gene expression or protein levels have utilized unvalidated reagents and assays, raising uncertainty about the interpretation of these results. We previously described a highly specific rabbit monoclonal antibody CRBN65 against 65-76 AA of human Cereblon. Here we describe a validated dual color bright-field Cereblon/CD138 immunohistochemical (IHC) assay utilizing CRBN65 and a commercial mouse monoclonal CD138 antibody. Sensitivity and specificity of the assay was determined and assay precision was shown for both cytoplasmic and nuclear Cereblon in MM bone marrow samples with coefficient of variation values of 5% and 2%, respectively. The dual IHC assay was effective for detecting a continuous range of Cereblon levels in 22 MM patient bone marrow core biopsies and aspirate clots, as shown by average cytoplasmic H-scores ranging from 63 to 267 and nuclear H-scores ranging from 17 to 250. Interpathologist comparison of MM sample H-scores by 3 pathologists demonstrated good concordance (R=0.73). This dual assay demonstrated superior Cereblon IHC measurement in MM samples compared with the single IHC assay using a published commercial rabbit polyclonal Cereblon antibody and could be used to explore the potential utility of Cereblon as a biomarker in the clinic.
Project description:Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.
Project description:Cereblon (CRBN) is a target for immunomodulatory drugs. This study investigated the prognostic value of the expression of CRBN-pathway genes on the clinical relevance of lenalidomide (Len) treatment and evaluated the levels of CRBN-binding proteins and mutations in these genes after Len treatment. Forty-eight primary multiple myeloma cells were collected prior to treatment with Len and dexamethasone (Ld) and 25 paired samples were obtained post-Ld therapy. These tumor cells were used to determine the expression and mutated forms of the CRBN-pathway genes. Following normalization with CRBN levels, there was a significantly reduced IKZF1/CRBN ratio in samples that responded poorly to Ld therapy. Moreover, patients with low ratios of IKZF1/CRBN showed a significantly shorter progression-free survival (PFS) and overall survival (OS) than those with higher ratios. However, patients with high ratios of KPNA2/CRBN showed a significantly shorter PFS and OS than patients with lower ratios. Of the 25 paired samples analyzed, most samples showed a reduction in the expression of CRBN and an increase in IKZF1 gene expression. No mutations were observed in CRBN, IKZF1, or CUL4A genes in the post-Ld samples. In conclusion, a decreased expression of IKZF1 and increased expression of KPNA2 compared to that of CRBN mRNA predicts poor outcomes of Ld therapy.
Project description:Lenalidomide (LEN) acts directly on multiple myeloma (MM) cells by inducing cereblon-mediated degradation of interferon regulatory factor 4, Ikaros (IKZF)1 and IKZF3, transcription factors that are essential for MM cell survival. The mucin 1 (MUC1) C-terminal transmembrane subunit (MUC1-C) oncoprotein is aberrantly expressed by MM cells and protects against reactive oxygen species (ROS)-mediated MM cell death. The present studies demonstrate that targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is more than additive with LEN in downregulating the WNT/?-catenin pathway, suppressing MYC, and inducing late apoptosis/necrosis. We show that the GO-203/LEN combination acts by synergistically increasing ROS and, in turn, suppressing ?-catenin. LEN resistance has been linked to activation of the WNT/?-catenin?CD44 pathway. In this regard, our results further demonstrate that targeting MUC1-C is effective against LEN-resistant MM cells. Moreover, GO-203 resensitized LEN-resistant MM cells to LEN treatment in association with suppression of ?-catenin and CD44. Targeting MUC1-C also resulted in downregulation of CD44 on the surface of primary MM cells. These findings, and the demonstration that expression of MUC1 and CD44 significantly correlate in microarrays from primary MM cells, provide support for combining GO-203 with LEN in the treatment of MM and in LEN-resistance.
Project description:Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.
Project description:Immunomodulatory drugs (IMiDs), including thalidomide derivatives such as lenalidomide and pomalidomide, offer therapeutic benefit in several hematopoietic malignancies and autoimmune/inflammatory diseases. However, it is difficult to study the IMiD mechanism of action in murine disease models because murine cereblon (CRBN), the substrate receptor for IMiD action, is resistant to some of IMiDs therapeutic effects. To overcome this difficulty, we generated humanized cereblon (CRBNI391V) mice thereby providing an animal model to unravel complex mechanisms of action in a murine physiological setup. In our current study, we investigated the degradative effect toward IKZF1 and CK-1?, a target substrate of IMiDs. Unlike WT mice which were resistant to lenalidomide and pomalidomide, T lymphocytes from CRBNI391V mice responded with a higher degree of IKZF1 and CK-1? protein degradation. Furthermore, IMiDs resulted in an increase in IL-2 among CRBNI391V mice but not in the WT group. We have also tested a thalidomide derivative, FPFT-2216, which showed an inhibitory effect toward IKZF1 protein level. As opposed to pomalidomide, FPFT-2216 and lenalidomide degrades CK-1?. Additionally, we assessed the potential therapeutic effects of IMiDs in dextran sodium sulfate (DSS)-induced colitis. In both WT and humanized mice, lenalidomide showed a significant therapeutic effect in the DSS model of colitis, while the effect of pomalidomide was less pronounced. Thus, while IMiDs' degradative effect on IKZF1 and CK-1?, and up-regulation of IL-2, is dependent on CRBN, the therapeutic benefit of IMiDs in a mouse model of inflammatory bowel disease occurs through a CRBN-IMiD binding region independent pathway.
Project description:Lenalidomide is a therapeutically active compound that binds to E3 ubiquitin ligase recruiter cereblon (CRBN) and induces cytotoxicity. We have identified eukaryotic translation initiation factor 2 subunit C2 (EIF2C2) as a new member of CRBN-downstream binding protein that plays an important role in microRNA (miRNA) maturation and function. The treatment of immunomodulatory drug (IMiD)-sensitive multiple myeloma (MM) cells with lenalidomide altered the steady-state levels of CRBN, EIF2C2 and miRNAs and induced apoptosis. However, although the treatment of IMiD-resistant MM cells with lenalidomide altered the steady-state levels of CRBN, EIF2C2 and miRNAs, but did not massively induce apoptosis. In contrast, silencing of EIF2C2 with its small hairpin RNA significantly altered the levels of miRNAs and induced apoptosis regardless of whether those cells are sensitive or resistant to IMiDs. Therefore, EIF2C2 could be considered as a new drug target for overcoming IMiDs resistance in MM cells. To find the role of EIF2C2 in MM cell growth, OCI-My5 cell lines My5/LV and My5/CRBN, with low and high CRBN expression, respectively, were treated 12 different ways. The steady-state levels of miRNAs between (1) My5/LV, EIF2C2-shRNA-treated My5/LV and EIF2C2-cDNA-treated My5/LV cells, (2) My5/CRBN, EIF2C2-shRNA-treated My5/CRBN and EIF2C2-cDNA-treated My5/CRBN cells, (3) My5/LV cells, My5/LV cells treated with 10 µM lenalidomide for 72 hours or 120 hours, and EIF2C2-cDNA-treated My5/LV cells treated with 10 µM lenalidomide for 72 hours, and (4) My5/CRBN cells, My5/CRBN cells treated with 10 µM lenalidomide for 72 hours or 120 hours, and EIF2C2-cDNA-treated My5/CRBN cells treated with 10 µM lenalidomide for 72 hours, were compared in this array.