Anthony W. Gebhard

Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases.

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009-0.0015 μM) from a single hit compound with weak inhibitory activity (IC50 = 15 μM), discovered by high-throughput screening. Structure-based design was performed using 35 cocrystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4, and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 μM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics.

Acquisition of resistance toward HYD1 correlates with a reduction in cleaved α4 integrin expression and a compromised CAM-DR phenotype.

We recently reported that the β1 integrin antagonist, referred to as HYD1, induces necrotic cell death in myeloma cell lines as a single agent using in vitro and in vivo models. In this article, we sought to delineate the determinants of sensitivity and resistance toward HYD1-induced cell death. To this end, we developed an HYD1 isogenic resistant myeloma cell line by chronically exposing H929 myeloma cells to increasing concentrations of HYD1. Our data indicate that the acquisition of resistance toward HYD1 correlates with reduced levels of the cleaved α4 integrin subunit. Consistent with reduced VLA-4 (α4β1) expression, the resistant variant showed ablated functional binding to fibronectin, VCAM-1, and the bone marrow stroma cell line HS-5. The reduction in binding of the resistant cell line to HS-5 cells translated to a compromised cell adhesion-mediated drug resistant phenotype as shown by increased sensitivity to melphalan- and bortezomib-induced cell death in the bone marrow stroma coculture model of drug resistance. Importantly, we show that HYD1 is more potent in relapsed myeloma specimens than newly diagnosed patients, a finding that correlated with α4 integrin expression. Collectively, these data indicate that this novel d-amino acid peptide may represent a good candidate for pursuing clinical trials in relapsed myeloma and in particular patients with high levels of α4 integrin. Moreover, our data provide further rationale for continued preclinical development of HYD1 and analogues of HYD1 for the treatment of multiple myeloma and potentially other tumors that home and/or metastasize to the bone. Mol Cancer Ther; 10(12); 2257–66. ©2011 AACR.

Emerging Strategies for Targeting Cell Adhesion in Multiple Myeloma

Multiple myeloma (MM) is an incurable hematological cancer involving proliferation of abnormal plasma cells that infiltrate the bone marrow (BM) and secrete monoclonal antibodies. The disease is clinically characterized by bone lesions, anemia, hypercalcemia, and renal failure. MM is presently treated with conventional therapies like melphalan, doxorubicin, and prednisone; or novel therapies like thalidomide, lenalidomide, and bortezomib; or with procedures like autologous stem cell transplantation. Unfortunately, these therapies fail to eliminate the minimal residual disease that remains persistent within the confines of the BM of MM patients. Mounting evidence indicates that components of the BM-including extracellular matrix, cytokines, chemokines, and growth factors-provide a sanctuary for subpopulations of MM. This co-dependent development of the disease in the context of the BM not only ensures the survival and growth of the plasma cells but contributes to de novo drug resistance. In addition, by fostering homing, angiogenesis, and osteolysis, this crosstalk plays a critical role in the progression of the disease. Not surprisingly then, over the past decade, several strategies have been developed to disrupt this communication between the plasma cells and the BM components including antibodies, peptides, and inhibitors of signaling pathways. Ultimately, the goal is to use these therapies in combination with the existing antimyeloma agents in order to further reduce or abolish minimal residual disease and improve patient outcomes.

MTI-101 (cyclized HYD1) binds a CD44 containing complex and induces necrotic cell death in multiple myeloma

Our laboratory recently reported that treatment with the d-amino acid containing peptide HYD1 induces necrotic cell death in multiple myeloma cell lines. Because of the intriguing biological activity and promising in vivo activity of HYD1, we pursued strategies for increasing the therapeutic efficacy of the linear peptide. These efforts led to a cyclized peptidomimetic, MTI-101, with increased in vitro activity and robust in vivo activity as a single agent using two myeloma models that consider the bone marrow microenvironment. MTI-101 treatment similar to HYD1 induced reactive oxygen species, depleted ATP levels, and failed to activate caspase-3. Moreover, MTI-101 is cross-resistant in H929 cells selected for acquired resistance to HYD1. Here, we pursued an unbiased chemical biology approach using biotinylated peptide affinity purification and liquid chromatography/tandem mass spectrometry analysis to identify binding partners of MTI-101. Using this approach, CD44 was identified as a predominant binding partner. Reducing the expression of CD44 was sufficient to induce cell death in multiple myeloma cell lines, indicating that multiple myeloma cells require CD44 expression for survival. Ectopic expression of CD44s correlated with increased binding of the FAM-conjugated peptide. However, ectopic expression of CD44s was not sufficient to increase the sensitivity to MTI-101–induced cell death. Mechanistically, we show that MTI-101–induced cell death occurs via a Rip1-, Rip3-, or Drp1-dependent and -independent pathway. Finally, we show that MTI-101 has robust activity as a single agent in the SCID-Hu bone implant and 5TGM1 in vivo model of multiple myeloma. Mol Cancer Ther; 12(11); 2446–58. ©2013 AACR.

Emerging Opportunities for Targeting the Tumor–Stroma Interactions for Increasing the Efficacy of Chemotherapy

It has become evident that tumor cells utilize survival signals that emanate from the tumor microenvironment to aid in survival and disease progression. Experimental evidence indicates that these same pathways contribute to de novo drug resistance. Identification of the mechanisms underlying the recruitment of accessory cells and survival signals provided by normal cells has provided a novel area for drug discovery for increasing the efficacy of cancer therapy.

Abstract 1523: α4 integrin expression is a determinant for activity of the integrin antagonist HYD1 in multiple myeloma cell lines and patient specimens

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Our laboratory previously showed that the β1 integrin inhibitory peptide HYD1 causes caspase independent cell death in myeloma cell lines. To further characterize the mechanism of HYD1 induced cell death an isogenic drug resistant variant was developed. This variant, called H929-60, was developed by chronically exposing parental H929 cells to increasing doses of HYD1 over time until a drug resistant phenotype was observed. The resistant cell lines demonstrated reduced binding of FAM-conjugated HYD1 to the cell surface compared to the parental cell line. We previously reported that HYD1 blocks α4β1 mediated adhesion to fibronectin and thus we asked whether resistance towards HYD1 induced cell death correlated with reduced α4β1 integrin expression. The resistant variant showed deceased levels of α4 integrin protein expression despite no change in RNA levels, indicating a post-transcriptional regulation of α4 expression. These changes were consistent with decreased binding to fibronectin, VCAM-1 and HS-5 stromal cells. Moreover, we demonstrated that biotin conjugated HYD1 is able to pulldown a α4 containing complex and the amount of α4 bound to biotin-HYD1 was reduced in the drug resistant variant. Reducing the expression of α4 and β1 integrins on the cell surface in H929 cells was sufficient to confer partial resistance to HYD1 induced cell death. Because the H929 resistant variant showed reduced adhesion to stroma cells, we asked whether acquisition of resistance towards HYD1 resulted in reduced levels of drug resistance in the HS-5 co-culture model of drug resistance. Using the HS-5 co-culture model, we show that the drug resistant variant H929-60 cells treated with either melphalan or velcade were drug sensitive in the HS-5 stromal co-culture model of drug resistance. Thus the drug resistant variant failed to demonstrate a cell adhesion mediate drug resistance (CAM-DR), indicating that as cells acquire resistance to HYD1 they lose resistance initiated by the tumor microenvironment. Finally, we show that HYD1 was significantly more potent in relapsed/refractory patient specimens compared to newly diagnosed patient specimens. Additionally, we show that α4 integrin expression positively correlates with HYD1 sensitivity. Together our data indicate that HYD1 is an attractive novel agent for the treatment of relapsed/refractory multiple myeloma with high levels of α4 integrin expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2011-1523

Abstract 3628: The forced interaction of α4 integrin and CD44 by the novel peptide HYD1 may be a determinant of HYD1-induced cell death in multiple myeloma

Our laboratory has previously shown that treatment with the novel d-amino acid peptide HYD1 (kikmviswkg) induces necrotic cell death in multiple myeloma (MM) cell lines. Furthermore, we have implicated the adhesion receptor VLA-4 as being involved in this process via shRNA silencing strategies. However, reducing α4 integrin only demonstrated partial protection indicating that other components of the HYD1 binding complex may be required for cell death. Thus, in the present study, we investigated the possible role of other adhesion molecules and/or complexes that may contribute to HYD1-induced cell death. Using an unbiased approach, we performed a total membrane pull-down with biotin conjugated HYD1 and NeutrAvidin beads on NCI-H929 MM cells. Samples were then loaded on an SDS-PAGE gel and analyzed by Mass Spectrometry. Data were then mined with the Scaffold 3 proteome software to determine pertinent hits. The adhesion receptor CD44 was determined to have the most peptide match hits and was confirmed as a valid hit via Western Blot analysis. Preliminary data using ELISA based binding studies with biotin-HYD1 as bait shows that recombinant CD44 directly binds to biotin-HYD1 in a concentration dependent manner. In order to identify complexes involved in HYD1-induced cell death, membrane immunoprecipitation assays were performed using CD44 and α4 integrin mAbs in both the presence and absence of HYD1. While Western Blot analysis confirmed basal levels of complex interactions between α4 integrin and CD44, the complex was significantly enhanced in the presence of HYD1. These data not only indicate a possible role of CD44 in HYD1-induced cell death, but also the ability of HYD1 to induce interactions among adhesion molecules and complexes. Further studies, including CD44shRNA targeting strategies, as well as looking at other adhesion molecules will be needed in order to confirm the role of individual cell surface receptors identified in the biotin-HYD1 complex in mediating HYD1 induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3628. doi:10.1158/1538-7445.AM2011-3628