Riggs, Daniel L.

Abstract The numerous and diverse genetic lesions in multiple myeloma (MM) make it difficult to discern those genetic abnormalities that may be suitable therapeutic targets. We have observed that the MYC locus is frequently rearranged in MM (about half of both patients and cell lines), suggesting that MYC dysregulation is a common feature of this disease. To evaluate MYC suppression as a therapeutic strategy in MM, we treated human myeloma cell lines with CPI203, a potent and selective inhibitor of bromodomain and extra-terminal (BET) proteins. The bromodomain inhibitor CPI203 has potency and selectivity similar to that of JQ1, but has improved pharmacokinetics for in vivo studies. From our collection of MM cell lines we have identified sensitive and resistant cell lines and characterized their response to CPI203 in detail. A number of conclusions have emerged from this work. First, response correlates with MYC protein levels after 3 days of treatment. MYC levels are significantly suppressed in sensitive lines and much less affected in resistant lines, supporting the hypothesis that MYC suppression alone determines cell fate in MM cell lines. In sensitive cell lines treatment with CPI203 caused a rapid (within 90 min.) decrease in MYC transcription that is consistent with the response being an intrinsic property of the cell lines. Inhibition of MYC transcription was followed by eventual cell cycle arrest and limited apoptosis in these sensitive cell lines. Both sensitive and resistant cell lines exhibited induction of Histone2H2BE gene in response to CPI203 treatment, indicating that drug efflux pumps alone do not mediate resistance. Surprisingly, several of the resistant cell lines exhibited an initial phase of sensitivity to BET inhibition. Upon drug addition, MYC transcription and MYC protein levels are repressed 2- to 5-fold, followed by a gradual recovery of MYC message and protein levels to the initial levels. In this second phase the cells are truly insensitive to BET inhibition: the drug remains active during this incubation and addition of fresh medium with drug does not affect MYC transcription. Furthermore, many aspects of the BET inhibition gene expression profile remain constant throughout this transition between sensitivity and resistance. These results suggest that the recovery phase is not a simple reversal of BET inhibition but rather the emergence of an alternative-signaling pathway that activates MYC expression in the presence of BET inhibition. Presently we are analyzing gene expression data to identify genes and signaling pathways responsible for this “induced resistance”. The identification of such pathways will help us better understand the molecular basis of the response to BET inhibition, which in turn might enable us to identify predictive biomarkers of the therapeutic response. Citation Format: Daniel L. Riggs, Marta Chesi, P Leif Bergsagel. Targeting MYC in multiple myeloma by BET protein inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2014-5489

Glioma cell migration correlates with Pyk2 activity, but the intrinsic mechanism that regulates the activity of Pyk2 is not fully understood. Previous studies have supported a role for the N-terminal FERM domain in the regulation of Pyk2 activity as mutations in the FERM domain inhibit Pyk2 phosphorylation. To search for novel protein-protein interactions mediated by the Pyk2 FERM domain, we utilized a yeast two-hybrid genetic selection to identify the mammalian Ste20 homolog MAP4K4 as a binding partner for the Pyk2 FERM domain. MAP4K4 coimmunoprecipitated with Pyk2 and was a substrate for Pyk2 but did not coimmunoprecipitate with the closely related focal adhesion kinase FAK. Knockdown of MAP4K4 expression inhibited glioma cell migration and effectively blocked Pyk2 stimulation of glioma cell. Increased expression of MAP4K4 stimulated glioma cell migration; however, this stimulation was blocked by knockdown of Pyk2 expression. These data support that the interaction of MAP4K4 and Pyk2 is integrated with glioma cell migration and suggest that inhibition of this interaction may represent a potential therapeutic strategy to limit glioblastoma tumor dispersion.

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

AbstractMolecular chaperones mediate multiple aspects of steroid receptor function, but the physiological importance of most receptor-associated cochaperones has not been determined. To help fill this gap, we targeted for disruption the mouse gene for the 52-kDa FK506 binding protein, FKBP52, a 90-kDa heat shock protein (Hsp90)-binding immunophilin found in steroid receptor complexes. A mouse line lacking FKBP52 (52KO) was generated and characterized. Male 52KO mice have several defects in reproductive tissues consistent with androgen insensitivity; among these defects are ambiguous external genitalia and dysgenic prostate. FKBP52 and androgen receptor (AR) are coexpressed in prostate epithelial cells of wild-type mice. However, FKBP52 and AR are similarly coexpressed in testis even though testis morphology and spermatogenesis in 52KO males are usually normal. Molecular studies confirm that FKBP52 is a component of AR complexes, and cellular studies in yeast and human cell models demonstrate that FKBP52 can enhance AR-meditated transactivation. AR enhancement requires FKBP52 peptidylprolyl isomerase activity as well as Hsp90-binding ability, and enhancement probably relates to an affect of FKBP52 on AR-folding pathways. In the presence of FKBP52, but not other cochaperones, the function of a minimally active AR point mutant can be dramatically restored. We conclude that FKBP52 is an AR folding factor that has critically important physiological roles in some male reproductive tissues.