Pharmacodynamic effects of the PARP inhibitor talazoparib (MDV3800, BMN 673) in patients with BRCA-mutated advanced solid tumors.

PURPOSE: Talazoparib is an inhibitor of the poly (ADP-ribose) polymerase (PARP) family of enzymes and is FDA-approved for patients with (suspected) deleterious germline BRCA1/2-mutated, HER2­negative, locally advanced or metastatic breast cancer. Because knowledge of the pharmacodynamic (PD) effects of talazoparib in patients has been limited to studies of PARP enzymatic activity (PARylation) in peripheral blood mononuclear cells, we developed a study to assess tumoral PD response to talazoparib treatment (NCT01989546). METHODS: We administered single-agent talazoparib (1 mg/day) orally in 28-day cycles to adult patients with advanced solid tumors harboring (suspected) deleterious BRCA1 or BRCA2 mutations. The primary objective was to examine the PD effects of talazoparib; the secondary objective was to determine overall response rate (ORR). Tumor biopsies were mandatory at baseline and post-treatment on day 8 (optional at disease progression). Biopsies were analyzed for PARylation, DNA damage response (γH2AX), and epithelial‒mesenchymal transition. RESULTS: Nine patients enrolled in this trial. Four of six patients (67%) evaluable for the primary PD endpoint exhibited a nuclear γH2AX response on day 8 of treatment, and five of six (83%) also exhibited strong suppression of PARylation. A transition towards a more mesenchymal phenotype was seen in 4 of 6 carcinoma patients, but this biological change did not affect γH2AX or PAR responses. The ORR was 55% with the five partial responses lasting a median of six cycles. CONCLUSION: Intra-tumoral DNA damage response and inhibition of PARP enzymatic activity were confirmed in patients with advanced solid tumors harboring BRCA1/2 mutations after 8 days of talazoparib treatment.

Combination therapy with pazopanib and tivantinib modulates VEGF and c-MET levels in refractory advanced solid tumors.

The vascular endothelial growth factor (VEGF)/VEGFR and hepatocyte growth factor (HGF)/c-MET signaling pathways act synergistically to promote angiogenesis. Studies indicate VEGF inhibition leads to increased levels of phosphorylated c-MET, bypassing VEGF-mediated angiogenesis and leading to chemoresistance. We conducted a phase 1 clinical trial with 32 patients with refractory solid tumors to evaluate the safety, pharmacokinetics, and pharmacodynamics of combinations of VEGF-targeting pazopanib and the putative c-MET inhibitor ARQ197 (tivantinib) at 5 dose levels (DLs). Patients either took pazopanib and tivantinib from treatment initiation (escalation phase) or pazopanib alone for 7 days, with paired tumor sampling, prior to starting combination treatment (expansion phase). Hypertension was the most common adverse event. No more than 1 dose limiting toxicity (DLT) occurred at any DL, so the maximum tolerated dose (MTD) was not determined; DL5 (800 mg pazopanib daily and 360 mg tivantinib BID) was used during the expansion phase. Twenty of 31 evaluable patients achieved stable disease lasting up to 22 cycles. Circulating VEGF, VEGFR2, HGF, and c-MET levels were assessed, and only VEGF levels increased. Tumor c-MET levels (total and phosphorylated) were determined in paired biopsies before and after 7 days of pazopanib treatment. Total intact c-MET decreased in 6 of 7 biopsy pairs, in contrast to previously reported c-MET elevation in response to VEGF inhibition. These results are discussed in the context of our previously reported analysis of epithelial-mesenchymal transition in these tumors.

Clinical Evolution of Epithelial-Mesenchymal Transition in Human Carcinomas.

The significance of the phenotypic plasticity afforded by epithelial-mesenchymal transition (EMT) for cancer progression and drug resistance remains to be fully elucidated in the clinic. We evaluated epithelial-mesenchymal phenotypic characteristics across a range of tumor histologies using a validated, high-resolution digital microscopic immunofluorescence assay (IFA) that incorporates ß-catenin detection and cellular morphology to delineate carcinoma cells from stromal fibroblasts and that quantitates the individual and colocalized expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (V) at subcellular resolution ("EMT-IFA"). We report the discovery of ß-catenin+ cancer cells that coexpress E-cadherin and vimentin in core-needle biopsies from patients with various advanced metastatic carcinomas, wherein these cells are transitioning between strongly epithelial and strongly mesenchymal-like phenotypes. Treatment of carcinoma models with anticancer drugs that differ in their mechanism of action (the tyrosine kinase inhibitor pazopanib in MKN45 gastric carcinoma xenografts and the combination of tubulin-targeting agent paclitaxel with the BCR-ABL inhibitor nilotinib in MDA-MB-468 breast cancer xenografts) caused changes in the tumor epithelial-mesenchymal character. Moreover, the appearance of partial EMT or mesenchymal-like carcinoma cells in MDA-MB-468 tumors treated with the paclitaxel-nilotinib combination resulted in upregulation of cancer stem cell (CSC) markers and susceptibility to FAK inhibitor. A metastatic prostate cancer patient treated with the PARP inhibitor talazoparib exhibited similar CSC marker upregulation. Therefore, the phenotypic plasticity conferred on carcinoma cells by EMT allows for rapid adaptation to cytotoxic or molecularly targeted therapy and could create a form of acquired drug resistance that is transient in nature. SIGNIFICANCE: Despite the role of EMT in metastasis and drug resistance, no standardized assessment of EMT phenotypic heterogeneity in human carcinomas exists; the EMT-IFA allows for clinical monitoring of tumor adaptation to therapy.

Novel antibody reagents for characterization of drug- and tumor microenvironment-induced changes in epithelial-mesenchymal transition and cancer stem cells.

The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.

Effective implementation of novel MET pharmacodynamic assays in translational studies.

MET tyrosine kinase (TK) dysregulation is significantly implicated in many types of cancer. Despite over 20 years of drug development to target MET in cancers, a pure anti-MET therapeutic has not yet received market approval. The failure of two recently concluded phase III trials point to a major weakness in biomarker strategies to identify patients who will benefit most from MET therapies. The capability to interrogate oncogenic mutations in MET via circulating tumor DNA (ctDNA) provides an important advancement in identification and stratification of patients for MET therapy. However, a wide range in type and frequency of these mutations suggest there is a need to carefully link these mutations to MET dysregulation, at least in proof-of-concept studies. In this review, we elaborate how we can utilize recently developed and validated pharmacodynamic biomarkers of MET not only to show target engagement, but more importantly to quantitatively measure MET dysregulation in tumor tissues. The MET assay endpoints provide evidence of both canonical and non-canonical MET signaling, can be used as "effect markers" to define biologically effective doses (BEDs) for molecularly targeted drugs, confirm mechanism-of-action in testing combination of drugs, and establish whether a diagnostic test is reporting MET dysregulation. We have established standard operating procedures for tumor biopsy collections to control pre-analytical variables that have produced valid results in proof-of-concept studies. The reagents and procedures are made available to the research community for potential implementation on multiple platforms such as ELISA, quantitative immunofluorescence assay (qIFA), and immuno-MRM assays.

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.

Inhibition of p38alpha MAPK disrupts the pathological loop of proinflammatory factor production in the myelodysplastic syndrome bone marrow microenvironment.

Myelodysplastic syndromes (MDS) are common causes of ineffective hematopoiesis and cytopenias in the elderly. Various myelosuppressive and proinflammatory cytokines have been implicated in the high rates of apoptosis and hematopoietic suppression seen in MDS. We have previously shown that p38 MAPK is overactivated in MDS hematopoietic progenitors, which led to current clinical studies of the selective p38alpha inhibitor, SCIO-469, in this disease. We now demonstrate that the myelosuppressive cytokines TNFalpha and IL-1beta are secreted by bone marrow (BM) cells in a p38 MAPK-dependent manner. Their secretion is stimulated by paracrine interactions between BM stromal and mononuclear cells and cytokine induction correlates with CD34+ stem cell apoptosis in an inflammation-simulated in vitro bone marrow microenvironment. Treatment with SCIO-469 inhibits TNF secretion in primary MDS bone marrow cells and protects cytogenetically normal progenitors from apoptosis ex vivo. Furthermore, p38 inhibition diminishes the expression of TNFalpha or IL-1beta-induced proinflammatory chemokines in BM stromal cells. These data indicate that p38 inhibition has anti-inflammatory effects on the bone marrow microenvironment that complements its cytoprotective effect on progenitor survival. These findings support clinical investigation of p38alpha as a potential therapeutic target in MDS and other related diseases characterised by inflammatory bone marrow failure.

Inhibition of the TGF-beta receptor I kinase promotes hematopoiesis in MDS.

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells, providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-beta-mediated gene activation in BM stromal cells, and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.

p38alpha-selective MAP kinase inhibitor reduces tumor growth in mouse xenograft models of multiple myeloma.

Multiple myeloma (MM) is a clonal plasma cell malignancy, which is currently incurable. Therefore, new mono- or combined therapy treatment regimens in the early and advanced phases of MM are urgently needed to combat this disease. Recently, p38 mitogen-activated protein kinase (MAPK) has been implicated as playing an important role in MM. Therefore, the effect of a p38alpha-selective MAPK inhibitor, SCIO-469 (indole-5-carboxamide, ATP-competitive inhibitor), or its structural analog, SD-282 (indole-5-carboxamide, ATP-competitive inhibitor) was examined in mouse xenograft models of MM using human RPMI-8226 or H-929 plasmacytoma inocula. Oral treatment with SCIO-469 (10, 30, 90 mg/kg) twice daily was initiated in mice with palpable tumors of RPMI-8226 origin, a condition that mimics early human myeloma disease. In mice with palpable tumors, 14 days of SCIO-469 treatment significantly reduced RPMI-8226 tumor growth in a dose-dependent manner. A significant dose-dependent reduction in RPMI-8226 tumor growth was also observed when SCIO-469 oral treatment at doses of 10, 30 and 90 mg/kg twice daily was initiated in mice with tumors of pronounced size, a condition that mimics advanced human myeloma disease. In a similar set of studies employing the SCIO-469 analogue SD-282 at 90 mg/kg/bid orally, histological assessment at the end of the study demonstrated a significant reduction in RPMI-8226 tumor growth and angiogenesis. SD-282 treatment was additionally shown to significantly reduced expression of heat-shock protein-27 (HSP-27) and phospho-p38 in the tumor cells. Furthermore, co-administration of SCIO-469 with dexamethasone elicited antitumor properties in dexamethasone-sensitive H-929 tumors at much lower than the typically effective doses of dexamethasone, suggesting its potential for combined therapy. In conclusion, p38 inhibitors reduced human myeloma cell growth in vivo both at early and advanced phases of the disease. The current study also provides evidence of potential for co-therapy with dexamethasone.

Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors.

The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38alpha by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38alpha isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS.

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation.

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.

Preventive and therapeutic potential of p38 alpha-selective mitogen-activated protein kinase inhibitor in nonobese diabetic mice with type 1 diabetes.

Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that function within T cells in both normal and stress-related pathophysiological states, including type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops T cell-mediated autoimmune pancreatic beta cell destruction that is similar to type 1 diabetes in humans. Because p38 MAPKs have been shown to modulate T cell function, we studied the effects of a p38alpha MAPK-selective inhibitor, indole-5-carboxamide (SD-169), on the development and progression of type 1 diabetes in the NOD mouse. In preventive treatment studies, SD-169 significantly reduced p38 and HSP60 expression in T cells of the pancreatic beta islets. Following treatment, the incidence of diabetes as determined by blood glucose levels was significantly lower, and immuno-histochemistry of pancreatic beta islet tissue demonstrated significant reduction in CD5+ T cell infiltration in the SD-169 treatment group as compared with untreated NOD mice. In therapeutic studies using mildly and moderately hyperglycemic NOD mice, SD-169 treatment lowered blood glucose and improved glucose homeostasis. Furthermore, following cessation of SD-169 treatment, NOD mice showed significant arrest of diabetes. In conclusion, we report that this p38alpha-selective inhibitor prevents the development and progression of diabetes in NOD mice by inhibiting T cell infiltration and activation, thereby preserving beta cell mass via inhibition of the p38 MAPK signaling pathway. These results have bearing on current prophylactic and therapeutic protocols using p38alpha-selective inhibitors in the prediabetic period for children at high risk of type 1 diabetes, in the honeymoon period, and for adults with latent autoimmune diabetes.

p38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells.

Although PS-341 (bortezomib) is a promising agent to improve multiple myeloma (MM) patient outcome, 65% of patients with relapsed and refractory disease do not respond. We have previously shown that heat shock protein (Hsp)27 is upregulated after PS-341 treatment, that overexpression of Hsp27 confers PS-341 resistance, and that inhibition of Hsp27 overcomes PS-341 resistance. Since Hsp27 is a downstream target of p38 mitogen-activated protein kinase (MAPK)/MAPK-mitogen-activated protein kinase-2 (MAPKAPK2), we hypothesized that inhibition of p38 MAPK activity could augment PS-341 cytotoxicity by downregulating Hsp27. Although p38 MAPK inhibitor SCIO-469 (Scios Inc, CA, USA) alone did not induce significant growth inhibition, it blocked baseline and PS-341-triggered phosphorylation of p38 MAPK as well as upregulation of Hsp27, associated with enhanced cytotoxicity in MM.1S cells. Importantly, SCIO-469 enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK) and augmented cleavage of caspase-8 and poly(ADP)-ribose polymerase. Moreover, SCIO-469 downregulated PS-341-induced increases in G2/M-phase cells, associated with downregulation of p21Cip1 expression. Importantly, SCIO-469 treatment augmented cytotoxicity of PS-341 even against PS-341-resistant cell lines and patient MM cells. These studies therefore provide the framework for clinical trials of SCIO-469 to enhance sensitivity and overcome resistance to PS-341, thereby improving patient outcome in MM.

APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA.

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-gamma. The induction of cytokine production by NK cells was preceded by activation of NF-kappaB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.