Richard Rickles

Adenosine A2A receptor agonists and PDE inhibitors: a synergistic multitarget mechanism discovered through systematic combination screening in B-cell malignancies.

Using a combination high-throughput screening technology, multiple classes of drugs and targeted agents were identified that synergize with dexamethasone (Dex) in multiple myeloma (MM) cells. Performing combination screening with these enhancers, we discovered an unexpected synergistic interaction between adenosine receptor agonists and phosphodiesterase (PDE) inhibitors that displays substantial activity in a panel of MM and diffuse large B-cell lymphoma (DLBCL) cell lines and tumor cells from MM patients. We have used selective adenosine receptor agonists, antagonists, and PDE inhibitors as well as small interfering RNAs targeting specific molecular isoforms of these proteins to dissect the molecular mechanism of this synergy. The adenosine A2A receptor and PDE2, 3, 4, and 7 are important for activity. Drug combinations induce cyclic AMP (cAMP) accumulation and up-regulate PDE4B. We also observe rigorous mathematical synergy in 3-way combinations containing A2A agonists, PDE inhibitors, and Dex at multiple concentrations and ratios. Taken together, these data suggest that A2A agonist/PDE inhibitor combinations may be attractive as an adjunctive to clinical glucocorticoid containing regiments for patients with MM or DLBCL and confer benefit in both glucocorticoid-sensitive and -resistant populations.

Adenosine A2A and Beta-2 Adrenergic Receptor Agonists: Novel Selective and Synergistic Multiple Myeloma Targets Discovered through Systematic Combination Screening

The use of combination drug regimens has dramatically improved the clinical outcome for patients with multiple myeloma. However, to date, combination treatments have been limited to approved drugs and a small number of emerging agents. Using a systematic approach to identify synergistic drug combinations, combination high-throughput screening (cHTS) technology, adenosine A2A and β-2 adrenergic receptor (β2AR) agonists were shown to be highly synergistic, selective, and novel agents that enhance glucocorticoid activity in B-cell malignancies. Unexpectedly, A2A and β2AR agonists also synergize with melphalan, lenalidomide, bortezomib, and doxorubicin. An analysis of agonists, in combination with dexamethasone or melphalan in 83 cell lines, reveals substantial activity in multiple myeloma and diffuse large B-cell lymphoma cell lines. Combination effects are also observed with dexamethasone as well as bortezomib, using multiple myeloma patient samples and mouse multiple myeloma xenograft assays. Our results provide compelling evidence in support of development of A2A and β2AR agonists for use in multi-drug combination therapy for multiple myeloma. Furthermore, use of cHTS for the discovery and evaluation of new targets and combination therapies has the potential to improve cancer treatment paradigms and patient outcomes. Mol Cancer Ther; 11(7); 1432–42. ©2012 AACR.

Identification of Combinatorial Drugs that Synergistically Kill both Eribulin-Sensitive and Eribulin- Insensitive Tumor Cells

Abstract Background: Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormally beating cilia. In these patients levels of nasal nitric oxide (nNO) are lower than those observed in healthy subjects. Objectives: We recorded the nNO levels in PCD patients in order to use those nNO measurements in the screening and identification of patients with symptoms suggestive of disease PCD disease.The microscopic cord formation is a characteristic property of the species of Mycobacterium tuberculosis complex (MTC). This feature is used as screening method of MTC and detection of drug resistant tuberculosis in law resource settings. The presence of true cording in M.abscessus poses a challenge for identification of MTC based on the cord formation.Background: Acute renal failure (ARF) continues to be a challenging problem in critically ill patients. We reviewed the nephrology consultations in our ICU to assess the necessity of those consultations and if there are any clinical criteria to indicate a necessary consultation.Eribulin sensitivity was examined in a panel of twenty-five human cancer cell lines representing a variety of tumor types, with a preponderance of breast and lung cancer cell lines. As expected, the cell lines vary in sensitivity to eribulin at clinically relevant concentrations. To identify combination drugs capable of increasing anticancer effects in patients already responsive to eribulin, as well as inducing de novo anticancer effects in non-responders, we performed a combinatorial high throughput screen to identify drugs that combine with eribulin to selectively kill tumor cells. Among other observations, we found that inhibitors of ErbB1/ErbB2 (lapatinib, BIBW-2992, erlotinib), MEK (E6201, trametinib), PI3K (BKM-120), mTOR (AZD 8055, everolimus), PI3K/mTOR (BEZ 235), and a BCL2 family antagonist (ABT-263) show combinatorial activity with eribulin. In addition, antagonistic pairings with other agents, such as a topoisomerase I inhibitor (topotecan hydrochloride), an HSP-90 inhibitor (17-DMAG), and gemcitabine and cytarabine, were identified. In summary, the preclinical studies described here have identified several combination drugs that have the potential to either augment or antagonize eribulin’s anticancer activity. Further elucidation of the mechanisms responsible for such interactions may be important for identifying valuable therapeutic partners for eribulin.

Abstract 5509: Identification of synergistic drugs using combination high-throughput screening

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Increased understanding of the complexity of biological networks is forcing us to reconsider traditional views of disease and treatment. This is particularly true for cancer, as tumor cells have a tremendous capacity to evade the effects of drugs by mutation and/or pathway adaptation. We have developed a combination high throughput screening (cHTS) platform (robotics and analytics) that systematically and efficiently identifies combination drugs with synergistic activity. Synergistic combination drugs can increase maximal drug effect, increase potency and/or circumvent chemoresistance. In addition, coordinated action at multiple molecular targets can provide unique therapeutic benefit not achievable with the “one-drug, one-target” paradigm, especially problematic for cancer. We describe the application of cHTS to the discovery of novel synergistic drug combinations for the treatment of cancer. Using cHTS we have made the surprising discovery that A2A and β2 adrenergic receptor agonists strongly synergize with glucocorticoids (GCs) to inhibit the proliferation of hematologic malignancies. Using an 83 cell line panel, we find that synergistic activity is specific for select B-cell malignancies (including some cell lines that are GC-insensitive). These dexamethasone-dependent synergies are observed across a large panel of multiple myeloma (MM) cell lines and in multiple complex preclinical models of MM disease. In general, chemoresistance is a recurrent problem for cancer drugs and development of resistance after chronic exposure can reduce drug efficacy and promote refractory disease. We therefore examined the effects of chronic exposure to either A2A or β2AR agonists. Exposure of MM cells (MM.1S) to CGS-21680 (A2A agonist) or salmeterol (β2AR agonist) for one month reduced single agent sensitivity >80%. Surprisingly, combinations of either agent with dexamethasone maintained similar amounts of synergy and cell killing as found with naive untreated cells. Our studies demonstrate that synergistic combinations of A2A and β2AR agonists are highly selective for B-cell malignancies and support the notion that synergistic drug combinations can improve therapeutically relevant selectivity and circumvent drug resistance. These preclinical studies reinforce the rationale for investigation of A2A and β2AR agonists in the treatment of B-cell malignancies and in particular, patients who have MM. Furthermore, this work highlights the power of cHTS to interrogate combination activity across large panels of cancer cell lines with distinct molecular phenotypes and to identify novel mechanisms for therapeutic application. 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 5509.

Abstract 4324: Predictive biomarker signatures for IAP inhibitor CUDC-427

The Inhibitors of Apoptosis (IAP) are a family of functionally related proteins that serve as endogenous regulators of apoptosis. The frequent overexpression of IAP proteins allows cancer cells to evade apoptosis and develop drug resistance, making them attractive targets for cancer therapies. CUDC-427 is a potent small molecule IAP antagonist currently tested in a Phase 1 clinical trial for solid tumors and lymphomas. Although only 10% of the cancer cell lines are sensitive to single-agent CUDC-427 in vitro, significant anti-tumor activity has been observed in xenograft models. These observations highlight the importance of identifying patients with sensitive tumors to better target CUDC-427 for specific patient populations. Previously, we have reported TNF family ligand induction and decrease in XIAP levels as potential predictive biomarkers for response to CUDC-427. However, this prediction method requires the detection of biomarker levels in post-treatment samples, which may not always be feasible in clinical setting. Therefore, we sought to assess predictive genetic markers of response to CUDC-427 treatment for patient stratification. Using an in vitro cell viability assay, single-agent activity of CUDC-427 was assessed against a panel of 90 hematological and solid tumor cell lines from the Cancer Cell Line Encyclopedia (CCLE) collection. These results and genomic data available from the CCLE database including gene expression, DNA copy number and mutation status were used in the elastic net analysis to identify predictors associated with drug response. A set of gene signatures containing about 10 significant predictors identified by this approach was further validated and refined in an independent set of 61 CCLE cell lines. The clinical relevance of these gene signatures was validated by searching the cBioPortal Cancer Genomics database. The results indicate that these signatures frequently occur in several solid and hematologic cancers. To further validate this set of gene signatures, single-agent activity of CUDC-427 was evaluated in 30 patient-derived xenograft tumor models and 12 cell-line-derived xenograft models including breast and ovarian cancers as well as lymphomas. Daily treatment with CUDC-427 induced 60% or greater tumor growth inhibition in 20 of the 42 models tested, including 4 highly sensitive models that demonstrated tumor regression. Whole genome data including mutation status, DNA copy number and mRNA expression profiles of these xenograft tumors were assessed to provide independent in vivo validation and to further refine the gene signatures generated in vitro. In conclusion, a set of gene signatures identified through this work can potentially be used as biomarkers to predict in vivo activity of CUDC-427 across multiple tumor types. These results will provide a basis for the development of predictive assays to aid selection of patients whose tumors may be sensitive to CUDC-427 treatment. Citation Format: Kaiming Sun, Ze Tian, Qi Zhang, Maria Samson, Ruzanna Atoyan, Mylissa Borek, Steven Dellarocca, Brian Zifcak, Troy Patterson, Anna W. Ma, Guangxin Xu, Michael J. Wick, Richard Rickles, Jing Wang. Predictive biomarker signatures for IAP inhibitor CUDC-427. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4324. doi:10.1158/1538-7445.AM2015-4324

Abstract 4765: Drug synergies observed for antibody and toxin components of SAR3419 ADC contribute to overall conjugate efficacy and can be combination drug or tumor cell line dependent

SAR3419, a novel antibody-drug conjugate (ADC), is composed of a humanized monoclonal IgG1 anti-CD19 antibody (huB4) attached by a cleavable linker to DM4, a derivative of cytotoxic agent maytansine (an inhibitor of microtubule polymerization). CD19 expression is restricted to B-cells and treatment of CD19-positive human lymphoma cells with SAR3419 can result in cell cycle arrest followed by apoptosis. As traditional monotherapies can rarely treat cancer effectively, particularly in non-solid tumours, a combination high throughput screen (cHTS) was performed where SAR3419 was combined with two hundred potential enhancer compounds across a panel of twenty cell lines using a 6x6 combination dose response matrix. The enhancer compounds compose a library of approved, emerging oncology drugs, and well-defined molecular probes. Robust synergies were observed between SAR3419 and PI3K inhibitors and examined further. Target/pathway inhibitor redundancy for the class of compounds served to validate PI3K inhibition as important for combination synergy. Surprisingly, for several of the cell lines, the molecular mechanism important for SAR3419-dependent combination activity is distinct. For example, for the Daudi and REC-1 tumor cell lines, PI3K inhibitor synergy can be attributed to the toxin DM4. In contrast for SU-DHL4 and SU-DHL6 cell lines, little combination activity is observed with toxin alone while the conjugate is strongly synergistic. By examining three-way combination activities (PI3K inhibitor x DM4 x huB4) using high resolution matrices, we show that the anti-CD19 antibody huB4 contributes to the synergy in the SU-DHL6 cell line and linkage of DM4 to huB4 (SAR3419) shifts both the potency and maximum activity for PI3K inhibitor combinations. The results demonstrate that the antibody component of an ADC can contribute to combination activity for specific drug pairings in a drug and cell-type specific manner and has wide ranging implications with respect to combination drug design. Citation Format: Richard J. Rickles, Thomas P. Giordano, Shakira F. Cotard, Jill M. Grenier, Angela Romanelli, Ti Cai. Drug synergies observed for antibody and toxin components of SAR3419 ADC contribute to overall conjugate efficacy and can be combination drug or tumor cell line dependent. [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 4765. doi:10.1158/1538-7445.AM2014-4765

Abstract 1698: Identification of drugs with eribulin combinatorial activity that kill both eribulin-sensitive and eribulin-insensitive tumor cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Eribulin mesylate, a nontaxane microtubule dynamics inhibitor with a mechanism distinct from most other antitubulin therapeutics, is clinically used in the United States for treatment of certain patients with locally advanced or metastatic breast cancer. Eribulin has broad anticancer activity in a wide variety of preclinical cancer models; as a result, numerous clinical trials are ongoing to investigate efficacy in other non-breast tumor types. We have examined eribulin sensitivity in a panel of 25 human cancer cell lines representing a variety of tumor types albeit with a preponderance of breast and lung cancer cell lines. As expected, the cell lines vary in sensitivity to eribulin at clinically relevant concentrations. To identify combination drugs capable of increasing anticancer effects in patients already responsive to eribulin, as well as inducing de novo anticancer effects in non-responders, we performed a combinatorial high throughput screen to identify drugs that combine with eribulin to selectively kill tumor cells. Among other observations, we found that inhibitors of ErbB1/ErbB2 (lapatinib, BIBW-2992, erlotinib), MEK (E6201, trametinib), PI3K (BKM-120), mTOR (AZD 8055, everolimus), and PI3K/mTOR (BEZ 235), and a BCL2 family antagonist (ABT-263) show combinatorial activity with eribulin. In addition, antagonistic pairings with other agents, such as topoisomerase I inhibitors (topotecan hydrochloride), an HSP-90 inhibitor (17-DMAG), and gemcitabine and cytarabine, were also identified. In summary, the preclinical studies described here have identified several combination drugs that have the potential to either augment or antagonize eribulins anticancer activity. Further elucidation of the mechanisms responsible for such interactions may be important for identifying valuable therapeutic partners for eribulin. Citation Format: Toshimitsu Uenaka, Richard Rickles, Yasuhiro Funahashi, Ping Zhu, Jill M. Grenier, Janine Steiger, Nanding Zhao, Bruce A. Littlefield, Junji Matsui, Kenichi Nomoto. Identification of drugs with eribulin combinatorial activity that kill both eribulin-sensitive and eribulin-insensitive tumor cells. [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 1698. doi:10.1158/1538-7445.AM2014-1698

Abstract 2215: Pharmacogenomic investigation of Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765): drug sensitivity in diffuse large B-cell lymphoma (DLBCL) within a tumor microenvironment-aligned high-throughput screen.

Ibrutinib (PCI-32765) is an orally administered small molecule that covalently binds to Cys-481 of Bruton9s tyrosine kinase (BTK). Ibrutinib has demonstrated promise across several types of B-cell malignancies and is currently in Phase 2/3 clinical testing. Preclinical studies have established three key mechanisms following blunting of proximal B cell receptor (BCR) signaling through BTK inhibition: (1) suppression of pro-survival pathways, (2) diminished integrin activation and (3) attenuation of chemotactic response. Single agent ibrutinib Phase 1/2 clinical trials have revealed high clinical response rates in both naive and relapsed or refractory chronic lymphocytic leukemia (CLL), as well as, relapsed or refractory mantle cell lymphoma (MCL) patients (Byrd, et al. 2012 ASH; Wang, et al. 2012 ASH; Advani, et al. 2010 JCO). Response rates in a phase 2 diffuse large B cell lymphoma (DLBCL) clinical trial appeared to be more prevalent in “activated B-cell” (ABC) over “germinal center B-cell like” (GCB) DLBCL patients (Staudt et al., ASH 2012). To gain a further understanding of ibrutinib response and inform optimal therapeutic combinations in DLBCL, we established a combination high throughput pharmacology screen (cHTS) with ibrutinib. Ibrutinib was evaluated alone and in combination with 99 targeted compounds, across 17 (12 GCB; 5 ABC) DLBCL cell lines. To better align with human biology, DLBCL cell lines were screened in the presence of human marrow stromal cell conditioned media (hMSC-CM) and B-cell receptor stimulation via anti-IgG/anti-IgM antibodies. Interestingly, 8/17 (47%) DLBCL cell lines were intolerant to any external BCR stimulation, so those lines were screened in hMSC-CM without exogenous BCR stimulation. Under our experimental conditions, 11/17 (65%) cell lines displayed some sensitivity (IC 50 TM measurements [breakdown: 4/17 (24%) highly sensitive (IC 50 50 200 nM 50 1 M 50 > 10 M)]. Consistent with DLBCL clinical responses, the highly sensitive (IC 50 Citation Format: Cuc Davis, Tineke Casneuf, Willem Lightenberg, Richard Rickles, Winnie Tam, Matthias Versele, Steven McClue, Sriram Balasubramanian, Joseph Buggy, Kate Sasser, Brett Hall. Pharmacogenomic investigation of Bruton9s tyrosine kinase (BTK) inhibitor ibrutinib (PCI-32765): drug sensitivity in diffuse large B-cell lymphoma (DLBCL) within a tumor microenvironment-aligned high-throughput screen. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2215. doi:10.1158/1538-7445.AM2013-2215