Chang-Xin Shi

Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-κB with antimyeloma activity in vitro and in vivo

As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-kappaB activation via inhibition of IKK alpha or IKK beta, whereas proteosome inhibitors instead suppress NF-kappaB function by impairing degradation of ubiquitinated I kappaB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-kappaB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-kappaB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC(50) < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib--providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-kappaB inhibitors as therapeutics for human multiple myeloma and related malignancies.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Identification of kinetin riboside as a repressor of CCND1 and CCND2 with preclinical antimyeloma activity

Knockout and transgenic studies in mice demonstrate that normal somatic tissues redundantly express 3 cyclin D proteins, whereas tumor cells seem dependent on a single overexpressed cyclin D. Thus, selective suppression of the individual cyclin D deregulated in a tumor represents a biologically valid approach to targeted cancer therapy. In multiple myeloma, overexpression of 1 of the cyclin D proteins is a ubiquitous feature, unifying at least 7 different initiating genetic events. We demonstrate here that RNAi of genes encoding cyclin D1 and cyclin D2 (CCND1 and CCND2, respectively) inhibits proliferation and is progressively cytotoxic in human myeloma cells. By screening a chemical library using a cell-based assay for inhibition of CCND2 trans-activation, we identified the plant cytokinin kinetin riboside as an inhibitor of CCND2 trans-activation. Kinetin riboside induced marked suppression of CCND2 transcription and rapidly suppressed cyclin D1 and D2 protein expression in primary myeloma cells and tumor lines, causing cell-cycle arrest, tumor cell-selective apoptosis, and inhibition of myeloma growth in xenografted mice. Mechanistically, kinetin riboside upregulated expression of transcription repressor isoforms of cAMP-response element modulator (CREM) and blocked both trans-activation of CCND2 by various myeloma oncogenes and cis-activation of translocated CCND1, suggesting induction of an overriding repressor activity that blocks multiple oncogenic pathways targeting cyclin D genes. These data support targeted repression of cyclin D genes as a therapeutic strategy for human malignancies.

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 Bla 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 (Th1) and T‐helper type 2 (Th2) 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. FASEBJ. 24, 947–956 (20l0). www.fasebj.org

Cereblon binding molecules in multiple myeloma.

Immunomodulation is an established treatment strategy in multiple myeloma with thalidomide and its derivatives lenalidomide and pomalidomide as its FDA approved representatives. Just recently the method of action of these cereblon binding molecules was deciphered and results from large phase 3 trials confirmed the backbone function of this drug family in various combination therapies. This review details the to-date knowledge concerning mechanism of IMiD action, clinical applications and plausible escape mechanisms in which cells may become resistant/refractory to cereblon binding molecule based treatment.

HSP90 inhibition drives degradation of FGFR2 fusion proteins: implications for treatment of cholangiocarcinoma

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F‐TKIs) can elicit meaningful objective clinical responses in patients carrying FF‐positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F‐TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2‐TACC3 (transforming acidic coiled‐coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2‐TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF‐driven ICC.

Abstract LB-128: Synthetic lethal RNAi screening identifies inhibition of Bcl-2 family members as sensitizers to 5-Azacytidine in myeloid cells

In order to identify novel targets and clinically exploitable vulnerabilities, we performed high-throughput RNA-interference (RNAi) synthetic lethality screening using myeloid cell lines TF-1 and ML-2 in combination with the hypomethylating agent 5-Azacytidine. A custom small interfering RNA (siRNA) library targeting nearly 1000 cancer-associated genes, including the human kinome, was utilized. Bcl-XL emerged as the strongest 5-Azacytidine sensitizing target in the erythroid cell line TF-1 but not in ML-2 cells. Additional siRNA sequences targeting Bcl-XL were transfected into a panel of myeloid cell lines and tested in drug dose response experiments using a ten-point serial dilution of 5-Azacytidine. The erythroid differentiated myeloid cell lines TF-1 and HEL both exhibited a 3 to 4-fold reduction in the IC50 of 5-Azacytidine upon knockdown of Bcl-XL, whereas IC50 values for non-erythroid, myeloid cell lines ML-2, THP-1 and MDS-L were unchanged. Furthermore, we tested lentiviral delivery of short-hairpin RNA (shRNA) targeting Bcl-XL in additional cell lines. Lentiviral knockdown of Bcl-XL confirmed 2 to 3-fold 5-Azacytidine sensitization in TF-1 and HEL cells while U937, HL-60, ML-2, THP-1 and MDS-L were not sensitized by Bcl-XL knockdown. Interestingly, while Bcl-XL knockdown with shRNA potentiated the effects of 5-Azacytidine in TF-1 and HEL, Bcl-XL knockdown alone resulted in considerable reductions in cell viability in TF-1 and HEL cells. These results demonstrate erythroid lineage-specific responses to Bcl-XL knockdown and suggest that Bcl-XL may represent an erythroid lineage-specific therapeutic vulnerability. To begin to explore this possibility we examined the putative BH3 mimetics Gossypol, Obatoclax and ABT-737 with 5-Azacytidine drug dose response experiments in myeloid cell lines. Neither Gossypol nor Obatoclax potentiated the effects of 5-Azacytdine. In striking contrast, ABT-737 resulted in a 2 to 4-fold reduction in the IC50 of 5-Azacytidine in all cell lines tested to date (TF-1, HEL, ML-2 and THP-1). ABT-737 sensitization to 5-Azacytidine was dose dependent. Furthermore, Combination Index (CI) values calculated with Calcusyn software demonstrate synergy between ABT-737 and 5-Azacytidine at clinically relevant concentrations. These results suggest that inhibition of anti-apoptotic Bcl-2 family members with a BH3 mimetic may represent an effective strategy for 5-Azacytidine combination therapy that can be directly translated into design of combination clinical trials in patients with myeloid leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-128.

Abstract C41: Chc1L is a tumor suppressor involved in development of histiocyte-rich neoplasms

Human chromosome 13q14 is one of the hotspots of chromosomal deletion in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. Chromosome Condensation 1-like ( CHC1L ) is an uncharacterized gene in this region. CHC1L is found within the smallest common region of loss of heterozygosity in prostate cancer, and its decreased expression is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. In the present study, we describe the generation and characterization of Chc1L gene knockout mice. Knockout mice develop normally and display an exaggerated proliferative response to LPS stimulation of cultured bone marrow and spleen cells at ten weeks of age. At approximately two years of age, both homozygous and heterozygous mice have markedly increased incidence of tumorigenesis, 80% and 56% respectively, compared to wild-type mice, 26%. The tumors are distributed most commonly to the spleen, mesenteric lymph nodes, liver and small intestine. The morphologic and immunophenotypic features of the tumor cells are consistent with those of tissue histiocytes, and most Chc1L knockout and heterozygote mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. These data provide the first direct evidence that Chc1L is a tumor suppressor gene involved in suppression of histiocyte-rich neoplasms. Citation Format: David Spillane, Ding Yan Wang, Changxin Shi, Youdong Wang, Anthony Gramolini, Keith Stewart, Susan Newbigging, Mingyao Liu, Xiao-Yan Wen. Chc1L is a tumor suppressor involved in development of histiocyte-rich neoplasms. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C41.