Apurva K. Srivastava

The National Cancer Institute ALMANAC: A Comprehensive Screening Resource for the Detection of Anticancer Drug Pairs with Enhanced Therapeutic Activity

To date, over 100 small-molecule oncology drugs have been approved by the FDA. Because of the inherent heterogeneity of tumors, these small molecules are often administered in combination to prevent emergence of resistant cell subpopulations. Therefore, new combination strategies to overcome drug resistance in patients with advanced cancer are needed. In this study, we performed a systematic evaluation of the therapeutic activity of over 5,000 pairs of FDA-approved cancer drugs against a panel of 60 well-characterized human tumor cell lines (NCI-60) to uncover combinations with greater than additive growth-inhibitory activity. Screening results were compiled into a database, termed the NCI-ALMANAC (A Large Matrix of Anti-Neoplastic Agent Combinations), publicly available at https://dtp.cancer.gov/ncialmanac Subsequent in vivo experiments in mouse xenograft models of human cancer confirmed combinations with greater than single-agent efficacy. Concomitant detection of mechanistic biomarkers for these combinations in vivo supported the initiation of two phase I clinical trials at the NCI to evaluate clofarabine with bortezomib and nilotinib with paclitaxel in patients with advanced cancer. Consequently, the hypothesis-generating NCI-ALMANAC web-based resource has demonstrated value in identifying promising combinations of approved drugs with potent anticancer activity for further mechanistic study and translation to clinical trials. Cancer Res; 77(13); 3564-76. ©2017 AACR.

Pharmacodynamic Response of the MET/HGF-Receptor to Small Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

Purpose: Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect. Experimental Design: Sandwich immunoassays and specimen handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models. Results: By limiting cold ischemia time to less than two minutes, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to 80% within 4 hours, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation. Conclusions: These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof of mechanism and proof of concept for the field. Clin Cancer Res; 22(14); 3683–94. ©2016 AACR.

Effect of a Smac Mimetic (TL32711, Birinapant) on the Apoptotic Program and Apoptosis Biomarkers Examined with Validated Multiplex Immunoassays Fit for Clinical Use

Purpose: To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors. Experimental Design: The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax–Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL–Bak heterodimer, Mcl1–Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis. Results: Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies. Conclusions: The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials. Clin Cancer Res; 22(4); 1000–10. ©2015 AACR.

Development and validation of a Luminex assay for detection of a predictive biomarker for PROSTVAC-VF therapy

Cancer therapies can provide substantially improved survival in some patients while other seemingly similar patients receive little or no benefit. Strategies to identify patients likely to respond well to a given therapy could significantly improve health care outcomes by maximizing clinical benefits while reducing toxicities and adverse effects. Using a glycan microarray assay, we recently reported that pretreatment serum levels of IgM specific to blood group A trisaccharide (BG-Atri) correlate positively with overall survival of cancer patients on PROSTVAC-VF therapy. The results suggested anti-BG-Atri IgM measured prior to treatment could serve as a biomarker for identifying patients likely to benefit from PROSTVAC-VF. For continued development and clinical application of serum IgM specific to BG-Atri as a predictive biomarker, a clinical assay was needed. In this study, we developed and validated a Luminex-based clinical assay for measuring serum IgM specific to BG-Atri. IgM levels were measured with the Luminex assay and compared to levels measured using the microarray for 126 healthy individuals and 77 prostate cancer patients. This assay provided reproducible and consistent results with low %CVs, and tolerance ranges were established for the assay. IgM levels measured using the Luminex assay were found to be highly correlated to the microarray results with R values of 0.93–0.95. This assay is a Laboratory Developed Test (LDT) and is suitable for evaluating thousands of serum samples in CLIA certified laboratories that have validated the assay. In addition, the study demonstrates that discoveries made using neoglycoprotein-based microarrays can be readily migrated to a clinical assay.

Molecular Pharmacodynamics-Guided Scheduling of Biologically Effective Doses: A Drug Development Paradigm Applied to MET Tyrosine Kinase Inhibitors

The development of molecularly targeted agents has benefited from use of pharmacodynamic markers to identify “biologically effective doses” (BED) below MTDs, yet this knowledge remains underutilized in selecting dosage regimens and in comparing the effectiveness of targeted agents within a class. We sought to establish preclinical proof-of-concept for such pharmacodynamics-based BED regimens and effectiveness comparisons using MET kinase small-molecule inhibitors. Utilizing pharmacodynamic biomarker measurements of MET signaling (tumor pY1234/1235MET/total MET ratio) in a phase 0–like preclinical setting, we developed optimal dosage regimens for several MET kinase inhibitors and compared their antitumor efficacy in a MET-amplified gastric cancer xenograft model (SNU-5). Reductions in tumor pY1234/1235MET/total MET of 95%–99% were achievable with tolerable doses of EMD1214063/MSC2156119J (tepotinib), XL184 (cabozantinib), and XL880/GSK1363089 (foretinib), but not ARQ197 (tivantinib), which did not alter the pharmacodynamic biomarker. Duration of kinase suppression and rate of kinase recovery were specific to each agent, emphasizing the importance of developing customized dosage regimens to achieve continuous suppression of the pharmacodynamic biomarker at the required level (here, ≥90% MET kinase suppression). The customized dosage regimen of each inhibitor yielded substantial and sustained tumor regression; the equivalent effectiveness of customized dosage regimens that achieve the same level of continuous molecular target control represents preclinical proof-of-concept and illustrates the importance of proper scheduling of targeted agent BEDs. Pharmacodynamics-guided biologically effective dosage regimens (PD-BEDR) potentially offer a superior alternative to pharmacokinetic guidance (e.g., drug concentrations in surrogate tissues) for developing and making head-to-head comparisons of targeted agents. Mol Cancer Ther; 17(3); 698–709. ©2018 AACR.

Abstract 5082: Impact of HGF knockin microenvironment on epithelial-mesenchymal transition and cancer stem cells in a non-small cell lung cancer xenograft model

We previously reported the generation of rabbit monoclonal antibodies to twelve EMT (epithelial-to-mesenchymal transition) transcription factors and cancer stem cell (CSC) markers for the development of pharmacodynamic assays to inform clinical trials of new anticancer therapies (Pfister et al., AACR 2013). Here we demonstrate the functional utility of some of these reagents in detecting HGF-induced changes in EMT and CSC biology in a xenograft tumor model. Initial antibody characterization was performed in vitro and a subset [including SNAIL, SLUG, SOX9, Goosecoid (GSC), NANOG and CD133] was selected for further testing of functional utility in FFPE tissues by quantitative multiplex IFA. The antibodies were applied to xenograft tissues derived from the non-small cell lung cancer tumor line, NCI-H596, implanted in hHGFscid/scid, hHGFki/scid or hHGFki/ki mice to examine HGF-induced changes in EMT factors, CSC markers, as well as pY1235-MET expression in vivo. H596 tumors grown in either hHGFki/scid or hHGFki/ki mice exhibited enhanced EMT particularly in tumor microenvironments adjacent to mouse stroma containing the HGF knockin gene, compared to those in hHGFscid/scid mice. By quantitative immunofluorescence, H596 tumors showed increased Vimentin:E-cadherin ratio when grown in hHGFki/scid (P Citation Format: Tony Navas, Thomas D. Pfister, Scott M. Lawrence, Apurva K. Srivastava, Robert J. Kinders, Suzanne Borgel, Sergio Alcoser, Melinda G. Hollingshead, Lindsay M. Dutko, Brad A. Gouker, Donna Butcher, Elinor Ng-Eaton, Naoko Takebe, Young H. Lee, Donald P. Bottaro, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow. Impact of HGF knockin microenvironment on epithelial-mesenchymal transition and cancer stem cells in a non-small cell lung cancer xenograft model. [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 5082. doi:10.1158/1538-7445.AM2015-5082

Abstract 1049: Quantitative immunofluorescence assessment of MET and epithelial to mesenchymal transition (EMT) biomarker modulation by antiangiogenic inhibitors in xenograft tumor tissues

We have previously reported the development of ELISA assays for intact MET, pY1235-MET, and pY1356-MET in tumor lysates (Srivastava et al, ASCO 2011). Here we report the development of a multiplex quantitative immunofluorescence assay (qIFA) for simultaneous detection of pY1235-MET and total MET in formalin-fixed paraffin-embedded (FFPE) tissues. We utilized a rabbit monoclonal antibody specific to pY1235-MET (with no detectable crossreactivity to pY1234-MET) to develop a highly specific and sensitive IFA assay with total MET (MET4 Mab; from G. Vande Woude, Van Andel Research Institute). Specificity of the IFA was demonstrated by HGF stimulation of A549 cells showing increased intensity of pY1235-MET which was completely inhibited by crizotinib but not by the non-MET specific multi-kinase inhibitor sorafenib, in vitro. The pY1235-MET epitope is stable in FFPE, and IFA detection was enhanced by EDTA (vs. Citrate) for antigen retrieval. Quantitative assessment of pY1235-MET was determined in xenograft models that demonstrate very high IFA expression of pY1235-MET and total MET via gene amplification (GTL-16 and SNU-5), lower expression in HGF-induced paracrine or autocrine cell lines (A549 and HT29), and no detectable staining for pY1235-MET in negative controls (SNU-1 and MDA-MB-231). We have demonstrated assay fitness for purpose using SNU-5 FFPE xenograft tumor samples from animals treated with increasing doses of crizotinib in vivo. Quantitative measurement by Definiens analysis of pY1235-MET and total MET in tissue regions of interest (ROI) showed 50% and 95% pY1235-MET inhibition with 12.5 mg/kg and 25 mg/kg crizotinib, respectively. There were no significant changes in total MET by IFA. A high correlation (R=0.899) was observed between % pY1235-MET/total MET ratio measured by IFA levels (expressed as the marker area/# nuclei for pY1235-MET and total MET per ROI) and % pY1235-MET/total MET ratio determined by ELISA levels (pM/ug protein). This quantitative MET IFA assay is currently being applied in conjunction with a previously developed EMT IFA assay (Navas et al, AACR 2013) for focal tissue analysis of pY1235-MET and total MET IFA expression as well as changes in epithelial to mesenchymal transition (EMT) in gastric tumor xenograft tissues treated with the VEGF inhibitor pazopanib, the MET inhibitor tivantinib (ARQ197), or the drug combination in vivo. These studies will determine whether pY1235-MET or total MET is induced by anti-VEGF inhibitors via increased EMT transition, and whether this effect can be reversed by combination with tivantinib. Funded by NCI Contract No. HHSN261200800001E Citation Format: Tony Navas, Scott L. Lawrence, Donna Butcher, Lindsay M. Dutko, Melinda G. Hollingshead, Robert J. Kinders, Ralph E. Parchment, Donald P. Bottaro, W. Marston Linehan, Joseph E. Tomaszewski, Apurva K. Srivastava, James H. Doroshow. Quantitative immunofluorescence assessment of MET and epithelial to mesenchymal transition (EMT) biomarker modulation by antiangiogenic inhibitors in xenograft tumor tissues. [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 1049. doi:10.1158/1538-7445.AM2014-1049

Abstract 3691: Met target inhibition-guided efficacy in preclinical models

Background: A direct comparison of drug efficacy for the multiple agents currently in clinical development targeting MET-driven cancers would be useful for the selection of optimal treatment options. Previously, we utilized validated MET pharmacodynamic (PD) assays to compare the time course of phosphorylated-MET (pMET) suppression for five MET inhibitors (ASCO 2013). In the current study, we selected three candidates that demonstrated potent MET inhibition to compare anti-tumor efficacy. Methods: PD time course and tumor PK data were utilized to simulate a dosing schedule anticipated to produce >90% pMET suppression in a SNU5 gastric cancer xenograft model. From these data, dosing schedules of 44 mg/kg (Q12H) cabozantinib, 12.5 mg/kg (Q12H) EMD1214063, and 16.5 mg/kg (QD) foretinib were chosen to achieve necessary tumor exposure to suppress pMET. Once tumors reached a 150±50 mm3 size, drugs were administered continuously for 21 days and tumor volumes were measured intermittently for 62 days. To measure pMET suppression, tumor quadrants were collected at 4, 12, and 24 hrs from all treatment groups after dose 1 (day 1) and on day 8 (after dose 8 of foretinib or dose 14 of cabozantinib and EMD1214063). Results: Intact MET levels (pM/μg protein) were approximately 70% lower than vehicle controls at all day 8 collection points for all three drugs. The pY1234/35MET/MET ratios were 89%-99% (p Conclusions: These studies demonstrate that PD response-guided regimens delivered drug doses that were lower than efficacious doses described previously (except cabozantinib), but effectively reduced tumor volume in SNU5 xenografts. The three MET inhibitors showed comparable anti-tumor efficacy when accompanied by equivalent pMET inhibition. Our data also affirms the utility of the MET PD assays to guide dose ranging studies. Funded by NCI Contract No HHSN261200800001E. Citation Format: Apurva K. Srivastava, Melinda G. Hollingshead, Jeevan P. Govindharajulu, Joseph M. Covey, Dane Liston, James Peggins, Donald P. Bottaro, John J. Wright, Robert J. Kinders, Joseph E. Tomaszewski, James H. Doroshow, Ralph E. Parchment. Met target inhibition-guided efficacy in preclinical models. [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 3691. doi:10.1158/1538-7445.AM2014-3691