Clifford J. Whatcott

Targeting the Tumor Microenvironment in Cancer: Why Hyaluronidase Deserves a Second Look

Increased extracellular matrix (ECM) deposition is a characteristic observed in many solid tumors. Increased levels of one ECM component-namely, hyaluronan (HA)-leads to reduced elasticity of tumor tissue and increased interstitial fluid pressure. Multiple initial reports showed that the addition of hyaluronidase (HYAL) to chemotherapeutic regimens could greatly improve efficacy. Unfortunately, the bovine HYAL used in those studies was limited therapeutically by immunologic responses to treatment. Newly developed recombinant human HYAL has recently been introduced into clinical trials. In this article, we describe the role of HA in cancer, methods of targeting HA, and clinical studies performed to date, and we propose that targeting HA could now be an effective treatment option for patients with many different types of solid tumors.

Inhibition of ROCK1 kinase modulates both tumor cells and stromal fibroblasts in pancreatic cancer

ROCK, or Rho-associated coiled coil-containing protein kinase, is a member of the AGC kinase family and has been shown to play a role in cell migration, ECM synthesis, stress-fiber assembly, and cell contraction. Increased ROCK expression has been reported in multiple pathological conditions, including cancer. Here, we report increased expression of ROCK 1 in pancreatic tumor epithelial cells as well as in cancer associated fibroblasts (CAF). In our analysis, 62% of tumor samples exhibited ≥2+ in staining intensity by IHC analysis, versus 40% of adjacent normal tissue samples (P<0.0001). Thus, we hypothesized that ROCKs may play a significant role in pancreatic cancer progression, and may serve as a suitable target for treatment. We report a low frequency (4/34) amplification of the ROCK1 gene locus at chromosome 18q11.1 in pancreatic ductal adenocarcinoma (PDAC) patient tissue samples by aCGH analysis. Inhibition of ROCK kinase activity by a small molecule inhibitor (fasudil) resulted in moderate (IC50s of 6–71 μM) inhibition of PDAC cell proliferation, migration, and activation of co-cultured stellate cells. In the KPC mouse model for pancreatic cancer, fasudil decreased tumor collagen deposition. This translated to an enhanced overall survival of the mice and an increase in gemcitabine uptake. Though fasudil may target both the tumor epithelial cells and the CAFs, our findings are consistent with the hypothesis that inhibition of tumor stroma enhances drug penetration and efficacy in PDAC. Overall, our data suggests that ROCK1 may serve as a potential therapeutic target to enhance current treatment regimens for pancreatic cancer.

Adenosquamous carcinoma of the pancreas: Molecular characterization of 23 patients along with a literature review

Adenosquamous carcinoma of the pancreas (ASCP) is a rare entity. Like adenocarcinoma of the pancreas, overall survival is poor. Characteristics of ASCP include central tumor necrosis, along with osteoclasts and hypercalcemia. Various theories exist as to why this histological subtype exists, as normal pancreas tissue has no benign squamous epithelium. Due to the rarity of this disease, limited molecular analysis has been performed, and those reports indicate unique molecular features of ASCP. In this paper, we characterize 23 patients diagnosed with ASCP through molecular profiling using immunohistochemistry staining, fluorescent in situ hybridization, chromogenic in situ hybridization, and gene sequencing, Additionally, we provide a comprehensive literature review of what is known to date of ASCP. Molecular characterization revealed overexpression in MRP1 (80%), MGMT (79%), TOP2A (75), RRM1 (42%), TOPO1 (42%), PTEN (45%), CMET (40%), and C-KIT (10%) among others. One hundred percent of samples tested were positive for KRAS mutations. This analysis shows heretofore unsuspected leads to be considered for treatments of this rare type of exocrine pancreas cancer. Molecular profiling may be appropriate to provide maximum information regarding the patients tumor. Further work should be pursued to better characterize this disease.

Abstract 235: AXL inhibition leads to a reversal of a mesenchymal phenotype sensitizing cancer cells to targeted agents and immuno-oncology therapies

Mesenchymal properties and the epithelial-to-mesenchymal transition (EMT) contribute to the initiation and progression of many tumor types and ultimately can lead to drug resistance and highly aggressive disease. It is becoming increasingly clear that the more mesenchymal characteristics cancer cells acquire the more resistant they become to standard chemotherapy, targeted agents, and even immune checkpoint inhibitors. We have been exploring the role of the receptor tyrosine kinase, AXL, and its related TAM family members, in promoting the mesenchymal phenotype in cancer cells and how these effects promote drug resistance and escape from immune surveillance. TP-0903, a potent AXL inhibitor, leads to a reversal of the mesenchymal phenotype in multiple cancer models. Following TP-0903 treatment, we observed changes in mRNA expression using RT-qPCR and protein expression using standard immunoblotting that are consistent with a reversal of the mesenchymal phenotype. Upon treatment with TP-0903 cancer cells possessed lower motility and a decrease in anchorage-independent growth, both hallmarks of a mesenchymal cell. In vivo models of erlotinib-resistant non-small cell lung cancer (NSCLC) were utilized to demonstrate TP-0903 single agent activity in highly mesenchymal models; however, more importantly, treatment with TP-0903 was able to sensitize this highly refractory model to erlotinib. AXL function and tumor mesenchymal characteristics also provide mechanisms for the cancer cells to evade immune surveillance. This is achieved by the role that AXL plays in detecting neighboring apoptotic cells resulting in the engulfment of dead cells (efferocytosis) and the associated debris in order to prevent the immune system9s exposure to auto-antigens under normal physiological conditions or exposure to cancer-associated neo-antigens in a tumor. Inhibition of AXL by TP-0903 can potentially inhibit tumor-associated efferocytosis leading to a stronger immunogenic response to the tumor. Indeed, results demonstrated synergy when TP-0903 was combined with an anti-PD-L1 agent in a syngeneic triple negative breast cancer mouse model. Interestingly, during the evaluation of TP-0903 in models of EMT, we detected dramatic change in the expression of the retinoic acid (RA) metabolizing protein CYP26A1, suggesting that AXL inhibition leads to changes in RA metabolism. Our data suggest that AXL induces a transition to a mesenchymal phenotype in cancer cells through the suppression of RA signaling and that TP-0903 can rapidly reverse this phenotype by signaling through RA causing the cell to revert to a more differentiated state. Due to its ability to reverse the aggressive mesenchymal phenotype of cancer cells, TP-0903 is a promising agent with the potential to have single agent activity and combined synergy with targeted anti-cancer agents and immunotherapies. Citation Format: Katherine K. Soh, Wontak Kim, Ye Sol Lee, Peter Peterson, Adam Siddiqui-Jain, Steven L. Warner, David J. Bearss, Clifford J. Whatcott. AXL inhibition leads to a reversal of a mesenchymal phenotype sensitizing cancer cells to targeted agents and immuno-oncology therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 235.

Abstract C202: CDK9 inhibition synergizes with BRD4 inhibitor-mediated super enhancer transcriptional repression in multiple preclinical tumor models

The group of transcriptional regulatory proteins known collectively as the super enhancer complex (SEC) coordinate the expression of entire genetic programs directing cell fate. The SEC is also important in driving cancer progression mediated by transcription of key oncogenes such as c-Myc and Bcl-2. The SEC requires the interaction and coordination of many proteins, including cyclin-dependent kinases (CDK), bromodomain proteins (BRD), histone deacetylases (HDAC), and histone methyltransferases (HMT). Each of these proteins are the focus of significant development efforts for the treatment of cancer. SEC-regulated transcription requires recruitment of CDK9/cyclin T1 from the 7SK RNA/Hexim1 inhibitory complex by BRD4 to transcriptional start sites. CDK9 then phosphorylates RNA polymerase II, releasing it from the start site leading to productive transcriptional elongation and gene expression. Considering the close association of CDK9 and BRD4, we hypothesized that the combination of CDK9 and BRD4 inhibitors would have synergistic effects in cancer cells. Alvocidib is a potent CDK9 inhibitor with validated clinical activity in AML from multiple Phase II studies in over 400 patients. Additionally, BRD4 inhibitors have demonstrated early promise in clinical studies with a focus on hematologic malignancies. However, we have found that CDK9 inhibitors, combined with bromodomain inhibitors, produced a synergistic effect by inhibiting the SEC more effectively than either of these compounds alone. Cell viability studies with various combinations resulted in an increase in potency. This was observed with alvocidib combined with JQ-1 (BRD4 inhibitor) in A549 lung cancer cells. Furthermore, the combination of alvocidib with JQ-1 completely abrogated SEC function, as measured by c-Myc or Mcl-1 expression through RT-qPCR. Similar results were achieved with other combinations of CDK9 and BRD4 inhibitors. These data, primarily focused on alvocidib and JQ-1, suggest a strong rationale for combining CDK9 and BRD4 inhibitors as a treatment strategy for multiple tumor types, including lung cancer. Furthermore, these findings may be more broadly applied to additional therapeutic targets in the SEC. These strategies yield synergistic effects at inhibiting SEC function and are highly active in tumor growth studies of cancer, in vivo. Clinical studies utilizing these combination strategies will explore this therapeutic approach. Citation Format: Ye Sol Lee, Wontak Kim, Katherine K. Soh, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. CDK9 inhibition synergizes with BRD4 inhibitor-mediated super enhancer transcriptional repression in multiple preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C202.

Abstract 2135: TGFβRI inhibition results in reduced collagen expression in pancreatic ductal adenocarcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic ductal adenocarcinoma (PDAC) is characterized by a robust stromal component termed the desmoplastic reaction. Transforming growth factor β (TGFβ) has been implicated in the activation of the cancer-associated fibroblast (CAF) that leads to pancreatic desmoplasia and the increased deposition of extracellular matrix proteins. Desmoplasia and its effects are thought to contribute to poor drug efficacy in PDAC treatment. We therefore hypothesized that TGFβ receptor (TGFβR) inhibition may serve to improve the activity of chemotherapeutics in PDAC by reducing the effects of desmoplasia. Using the TGFβRI inhibitors, LY2157299 and SB431542, we assessed the effects of TGFβR inhibition on cell proliferation and collagen production in both mono- and co-cultured pancreatic tumor cells and CAFs. We found that although the TGFβRI inhibitors had moderate effect on the proliferation of PDAC cells and co-cultured CAF cells, they significantly inhibited collagen 1 expression in the co-culture of MIA PaCa-2 and SU86.86 cells with CAFs (2.3 and 6.0-fold at the mRNA level, respectively). Similar qualitative reductions were also observed in immunofluorescence microscopy and immunoblots probing for collagen 1 protein levels. We also sought to determine if TGFβR inhibition would result in a reduction in collagen expression in the more biologically relevant in vivo model for PDAC, the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mouse. KPC mice were treated orally, b.i.d., with 75mg/kg LY2157299 and by i.p., with gemcitabine (q3dx4) at 80mg/kg. Tumors in KPC mice treated with the combination of LY2157299 and gemcitabine showed a marked reduction in overall stromal content when compared to vehicle-treated mice. Our results suggest that TGFβR inhibition may be an effective means of ameliorating the effects of desmoplasia in PDAC, which may in turn improve the efficacy of current PDAC treatment regimens. Citation Format: Clifford J. Whatcott, Sabrina N. Dumas, Aprill Watanabe, Janine LoBello, Daniel D. Von Hoff, Haiyong Han. TGFβRI inhibition results in reduced collagen expression in pancreatic ductal adenocarcinoma. [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 2135. doi:10.1158/1538-7445.AM2013-2135

Parallel Accumulation of Tumor Hyaluronan, Collagen and Other Drivers of Tumor Progression

Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors. Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance. Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content. Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798–807. ©2018 AACR.

Abstract 1106: Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma

The proteasome inhibitor bortezomib is widely used in the treatment of patients with multiple myeloma (MM). The expression levels of many proteins increase as a result of bortezomib treatment, including the pro-apoptotic protein NOXA. NOXA functions to sequester the anti-apoptotic BCL-2 family member, MCL-1. High levels of MCL-1 and/or low levels of NOXA have been implicated in bortezomib resistance and negative patient outcomes, including short duration of treatment response. The BCL-2-specific BH3 mimetic venetoclax (ABT-199) has also been explored in multiple hematological malignancies, including the treatment of MM. Venetoclax induces apoptosis in a BCL-2 specific manner by directly inhibiting BCL-2 function. However, intrinsic resistance to venetoclax treatment observed in MM patient samples has been attributed to a low BCL-2-to-MCL-1 gene expression ratio, suggesting a central role for MCL-1 in cell survival in this context as well. Increased MCL-1 expression is a known resistance mechanism to venetoclax treatment in a variety of cell types including chronic lymphocytic leukemia and lymphomas. Considering the central role of MCL-1 to treatment efficacy in MM, we investigated the ability of an MCL-1-lowering agent, namely the CDK9 inhibitor alvocidib, to potentiate the activity of venetoclax in MM. Alvocidib suppresses MCL-1 expression via CDK9-mediated regulation of RNA polymerase II. Alvocidib has achieved robust improvements in the clinical response rates of high-risk, newly diagnosed acute myeloid leukemia (AML) patients as part of the time-sequential ACM regimen (alvocidib + cytarabine + mitoxantrone). We therefore hypothesized that alvocidib would potentiate the activity of venetoclax in MM through an MCL-1-dependent mechanism. In this report, we demonstrate that alvocidib inhibits the protein expression of MCL-1 in MM cells in a time-dependent fashion, up to 96 hours. In cell viability assays, the addition of up to 100 nM venetoclax resulted in a 2.8-fold reduction in the IC50 of alvocidib in the cultured OPM-2 cell line. Conversely, the potentiation of venetoclax activity with the addition of alvocidib resulted in a more than 500-fold decrease in IC50 in the relatively venetoclax-resistant OPM-2 cells. Additional studies are currently underway to investigate the efficacy of alvocidib and venetoclax in the context of bortezomib resistance where low NOXA may contribute to enhanced cell survival via MCL-1. Taken together, our data suggest that the combination of alvocidib with venetoclax may constitute a novel therapeutic regimen in the treatment of MM. Further, it suggests that CDK9-mediated targeting of MCL-1 may offer a clinical route to addressing intrinsic resistance in MM patients. Citation Format: Mark Livingston, Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2017-1106

Abstract 5133: TP-1287, an oral prodrug of the cyclin-dependent kinase-9 inhibitor alvocidib

Alvocidib is a potent inhibitor of cyclin-dependent kinase-9 (CDK9) and induces apoptosis in cancer cells by reducing the expression of short-lived, anti-apoptotic proteins such as MCL-1. Alvocidib, as a part of a sequential combination regimen with cytarabine and mitoxantrone (ACM), is currently in a Phase II clinical trial in relapsed/refractory acute myeloid leukemia (AML). Patients with AML that have a high dependence on MCL-1 are considered more likely to benefit from the alvocidib-containing regimen. MCL-1 has emerged as a key protein in drug resistance of multiple solid tumor types including breast, prostate and lung cancers. The use of alvocidib in clinical settings beyond the ACM regimen is somewhat limited by the current intravenous route of administration. An orally administered form of alvocidib would allow prolonged repression of MCL-1 through chronic dosing and scheduling. Alvocidib itself is highly permeable in CACO-2 monolayers and is soluble at acidic pHs but solubility is strikingly reduced at neutral or basic conditions, which could hamper the development of an oral formulation. We hypothesized that a phosphate prodrug of alvocidib would improve solubility under neutral or basic conditions and enable the efficient systemic delivery of alvocidib via oral administration. We synthesized a phosphate prodrug of alvocidib, TP-1287, in three steps from the parent compound. The solubility of TP-1287, was determined at various pH levels. It was found to be highly soluble under acidic, neutral, and basic conditions (1.5 mg/mL at pH 2.2; 1.8 mg/mL at pH 4.5; 9.5 mg/mL at pH 6.8 and 9.3 mg/mL at pH 8.7) compared to alvocidib (4.4 mg/mL at pH 2.2; 1.3 mg/mL at pH 4.5; 0.02 mg/mL at pH 6.8 and 0.02 mg/mL at pH 8.7). Pharmacokinetic studies were conducted in mice in which TP-1287 was efficiently converted to the parent alvocidib (Cmax = 1922.7 ng/ml, t1/2 = 4.4 hr) with high oral bioavailability (%F = 182.3, compared to intravenous alvocidib). Efficacy and pharmacodynamic studies (measuring MCL-1 expression levels), were evaluated in tumor xenograft models. TP-1287 demonstrated significant anti-tumor efficacy in the MV4-11 AML mouse xenograft model and produced as much as a 61.7% inhibition of the pharmacodynamic biomarker MCL-1 in xenografted tumors, demonstrating a wide, 75-fold therapeutic dosing window. In addition, TP-1287 strongly inhibited tumor growth, achieving 109.1% tumor growth inhibition (%TGI) at the 7.5 mg/kg dose level. TP-1287 is highly soluble over a broader pH range than alvocidib and is efficiently metabolized to the parent compound in vivo, following oral administration. Tumor xenograft models and pharmacodynamic studies indicate that oral delivery of TP-1287 is efficacious in mice. Based on these results, we anticipate moving TP-1287, as an orally delivered CDK9 inhibitor, into a forthcoming clinical trial directed towards solid tumors vulnerable to the suppression of MCL-1. Citation Format: Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Steven Weitman, Steven L. Warner, David J. Bearss, Adam Siddiqui-Jain. TP-1287, an oral prodrug of the cyclin-dependent kinase-9 inhibitor alvocidib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5133. doi:10.1158/1538-7445.AM2017-5133

Abstract IA29: Identifying epithelial morphogenesis inhibitors in neural crest development and cancer

The epithelial to mesenchymal transition (EMT) is a highly conserved morphogenesis program that is essential for re-shaping and mobilizing epithelial cells during gastrulation, neural crest development and tissue regeneration. In cancer cells, EMT induction promotes acquisition of invasive cellular morphologies, stem cell-like properties and pro-survival mechanisms, which contribute to disease progression, therapy resistance and decreased overall survival. The identification of compounds that block or reverse EMT therefore represents an important therapeutic strategy to prevent cancer invasion and eradicate disseminated tumor cells. Unfortunately, current EMT inhibitors have shown limited clinical benefit, in part due to an incomplete understanding of the molecular mechanisms controlling EMT in development and cancer as well as a lack of screening platforms that recapitulate the complex physiological environment of EMT in the living animal. To overcome these obstacles, we have established a zebrafish Snail1-GFP lineage reporter strain to label dorsal neural tube progenitor cells before they undergo EMT to become neural crest, which allows us to visualize epithelial morphogenesis independent of later cell migration events. Thousands of Snail1-GFP embryos can be easily generated to perform whole animal-based screens with small molecule libraries to identify compounds that inhibit EMT in vivo. Our initial screening results using previously characterized EMT inhibitors showed that many compounds inhibited neural crest migration after EMT had occurred, but only one compound, an AXL receptor tyrosine kinase inhibitor called TP-0903, inhibited both epithelial morphogenesis and neural crest migration. TP-0903 significantly decreased twist1a expression, a canonical EMT transcription factor and blocked down-regulation of epithelial Cadherins. RNA-Seq analysis and chemical rescue experiments revealed that TP-0903 acts by inducing retinoic acid (RA) biosynthesis and triggering a RA-mediated transcriptional program. TP-0903 treatment of a number of human cell lines and mouse xenograph tumors showed that TP-0903 also inhibits EMT programs and survival in human cancer cells. These studies demonstrate the value and feasibility of using zebrafish neural crest development to identify effective EMT compounds in vivo. As such, we have identified TP-0903 as a new potential therapeutic for inhibiting EMT in cancer, and our findings support the hypothesis that RA-induced inhibition of EMT contributes to its current success in treating minimal residual disease in humans. Citation Format: Laura Jimenez, Clifford Whatcott, Jindong Wang, Steven Warner, Monique Morrison, David Bearss, Rodney A. Stewart. Identifying epithelial morphogenesis inhibitors in neural crest development and cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA29.