Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes.

In this study, targeted sequencing to screen 50 multidrug refractory multiple myeloma (rMM) patients was performed by using the Multiple Myeloma Mutation Panel. Patients were pretreated with both immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs), and 88%, 78%, and 68% were refractory to an IMiD, a PI, or both, respectively. The majority of patients had progressive (82%) or refractory (78%) disease immediately before sampling, with 43% being IMiD refractory and 46% being PI refractory in the most recent line of therapy. Compared with newly diagnosed MM, an increased prevalence of mutations in the Ras pathway genes KRAS, NRAS, and/or BRAF (72%), as well as TP53 (26%), CRBN (12%), and CRBN pathway genes (10%) was observed. Longitudinal analyses performed in 3 patients with CRBN mutations at time of IMiD resistance confirmed that these mutations were undetectable at earlier, IMiD-sensitive time points. Furthermore, the functional introduction of these mutations in MM cells conferred lenalidomide resistance in vitro. These data indicate a differential genetic landscape in rMM associated with drug response.

Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival.

BACKGROUND: Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity. METHODS: Cereblon binding partners were identified from a MM cell line expressing histidine-tagged cereblon by pulling down cereblon and its binding partners and verified by co-immunoprecipitation. IMiD effects were determined by western blot analysis, cell viability assay, microRNA array and apoptosis analysis. RESULTS: We identified argonaute 2 (AGO2) as a cereblon binding partner and found that the steady-state levels of AGO2 were regulated by cereblon. Upon treatment of IMiD-sensitive MM cells with lenalidomide, the steady-state levels of cereblon were significantly increased, whereas levels of AGO2 were significantly decreased. It has been reported that AGO2 plays a pivotal role in microRNA maturation and function. Interestingly, upon treatment of MM cells with lenalidomide, the steady-state levels of microRNAs were significantly altered. In addition, silencing of AGO2 in MM cells, regardless of sensitivity to IMiDs, significantly decreased the levels of AGO2 and microRNAs and massively induced cell death. CONCLUSION: These results support the notion that the cereblon binding partner AGO2 plays an important role in regulating MM cell growth and survival and AGO2 could be considered as a novel drug target for overcoming IMiD resistance in MM cells.

Mechanism of immunomodulatory drugs' action in the treatment of multiple myeloma.

Although immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, are widely used in the treatment of multiple myeloma (MM), the molecular mechanism of IMiDs' action is largely unknown. In this review, we will summarize recent advances in the application of IMiDs in MM cancer treatment as well as their effects on immunomodulatory activities, anti-angiogenic activities, intervention of cell surface adhesion molecules between myeloma cells and bone marrow stromal cells, anti-inflammatory activities, anti-proliferation, pro-apoptotic effects, cell cycle arrest, and inhibition of cell migration and metastasis. In addition, the potential IMiDs' target protein, IMiDs' target protein's functional role, and the potential molecular mechanisms of IMiDs resistance will be discussed. We wish, by presentation of our naive discussion, that this review article will facilitate further investigation in these fields.

Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.

Hacs1, a SH3 and SAM domain-containing adaptor protein, is up-regulated by IL-4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1(-/-) mice by deletion of the SH3 and SAM domains. Hacs1(-/-) mice were viable and fertile and had normal bone marrow B-cell development and normal splenic T- and B-cell populations. However, adult Hacs1(-/-) mice had increased peritoneal B1a cells (IgM(+)CD5(+)). On immunization with T-cell-independent antigen TNP-Ficoll, Hacs1(-/-) mice had increased production of anti-TNP IgM and IgG3. Purified splenic B cells from Hacs1(-/-) mice showed increased cell proliferation on BCR (B-cell receptor) stimulation. We further demonstrate that the Hacs1(-/-) B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T-helper type 1 (T(h)1) and T-helper type 2 (T(h)2) humoral responses were enhanced in Hacs1(-/-) mice. In vitro bone marrow-derived Hacs1(-/-) dendritic cells showed increased IL-12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.-Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.-Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.

Cyproheptadine displays preclinical activity in myeloma and leukemia.

D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G(0)/G(1) phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drug's known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer.

A chemical biology screen identifies glucocorticoids that regulate c-maf expression by increasing its proteasomal degradation through up-regulation of ubiquitin.

The oncogene c-maf is frequently overexpressed in multiple myeloma cell lines and patient samples and contributes to increased cellular proliferation in part by inducing cyclin D2 expression. To identify regulators of c-maf, we developed a chemical screen in NIH3T3 cells stably overexpressing c-maf and the cyclin D2 promoter driving luciferase. From a screen of 2400 off-patent drugs and chemicals, we identified glucocorticoids as c-maf-dependent inhibitors of cyclin D2 transactivation. In multiple myeloma cell lines, glucocorticoids reduced levels of c-maf protein without influencing corresponding mRNA levels. Subsequent studies demonstrated that glucocorticoids increased ubiquitination-dependent degradation of c-maf and up-regulated ubiquitin C mRNA. Moreover, ectopic expression of ubiquitin C recapitulated the effects of glucocorticoids, demonstrating regulation of c-maf protein through the abundance of the ubiquitin substrate. Thus, using a chemical biology approach, we identified a novel mechanism of action of glucocorticoids and a novel mechanism by which levels of c-maf protein are regulated by the abundance of the ubiquitin substrate.

Promiscuous mutations activate the noncanonical NF-kappaB pathway in multiple myeloma.

Activation of NF-kappaB has been noted in many tumor types, however only rarely has this been linked to an underlying genetic mutation. An integrated analysis of high-density oligonucleotide array CGH and gene expression profiling data from 155 multiple myeloma samples identified a promiscuous array of abnormalities contributing to the dysregulation of NF-kappaB in approximately 20% of patients. We report mutations in ten genes causing the inactivation of TRAF2, TRAF3, CYLD, cIAP1/cIAP2 and activation of NFKB1, NFKB2, CD40, LTBR, TACI, and NIK that result primarily in constitutive activation of the noncanonical NF-kappaB pathway, with the single most common abnormality being inactivation of TRAF3. These results highlight the critical importance of the NF-kappaB pathway in the pathogenesis of multiple myeloma.

Soluble interleukin-13Ralpha2 decoy receptor inhibits Hodgkin's lymphoma growth in vitro and in vivo.

Recent studies have demonstrated that the malignant Reed-Sternberg cells of Hodgkin's lymphoma (HL) secrete and are responsive to interleukin (IL)-13. We hypothesized that overexpression of a soluble IL-13 decoy receptor (sIL-13Ralpha2) via adenoviral-mediated gene transfer would inhibit IL-13-induced Reed-Sternberg cell proliferation. Western blot and ELISA analysis verified expression of sIL-13Ralpha2 in cell lysates and supernatants of AdsIL-13Ralpha2-transduced COS-7 cells. Treatment of two IL-13-responsive HL-derived cell lines, HDLM-2 and L-1236, with AdsIL-13Ralpha2-conditioned medium, resulted in the inhibition of cell proliferation, and down-regulated the phosphorylation of signal transducer and activator of transcription 6 (STAT6), an important mediator of IL-13 signaling. i.v. delivery of AdsIL-13Ralpha2 in NOD/SCID mice with s.c. implanted HDLM-2 cells delayed tumor onset and growth while enhancing survival compared with control mice. Intratumoral administration of AdsIL-13Ralpha2 led to the regression or stabilization of established tumors and was associated with diminished STAT6 phosphorylation. Our data demonstrate that AdsIL-13Ralpha2 can suppress HL growth in vitro and in vivo.

Specific homeodomain-DNA interactions are required for HOX11-mediated transformation.

HOX11 encodes a homeodomain protein that is aberrantly expressed in T-cell acute lymphoblastic leukemia as a consequence of the t(10;14) and t(7;10) chromosomal translocations. We previously reported that HOX11 immortalizes murine hematopoietic progenitors and induces pre-T-cell tumors in mice after long latency. It has been demonstrated in a number of studies that HOX11, similar to other homeodomain proteins, binds DNA and transactivates transcription. These findings suggest that translocation-activated HOX11 functions as an oncogenic transcription factor. Here we report that HOX11 represses transcription through both TATA-containing and TATA-less promoters. Interestingly, transcriptional repression by HOX11 is independent of its DNA binding capability. Moreover, a systematic mutational analysis indicated that repressor activity was separable from immortalizing function, which requires certain residues within the HOX11 homeodomain that make base-specific or phosphate-backbone contacts with DNA. We further showed that the pathologic action of HOX11 involves DNA binding-dependent transcriptional pathways that are distinct from those controlling expression of a chromosomal target gene (Aldh-1). We conclude that dysregulated expression of a particular set of downstream target genes by DNA binding via the homeodomain is of central importance for leukemia initiation mediated by HOX11.