Sundeep Dugar

Targeting Endogenous Transforming Growth Factor β Receptor Signaling in SMAD4-Deficient Human Pancreatic Carcinoma Cells Inhibits Their Invasive Phenotype 1

Transforming growth factor-β (TGF-β) suppresses tumor formation by blocking cell cycle progression and maintaining tissue homeostasis. In pancreatic carcinomas, this tumor suppressive activity is often lost by inactivation of the TGF-β-signaling mediator, Smad4. We found that human pancreatic carcinoma cell lines that have undergone deletion of MADH4 constitutively expressed high endogenous levels of phosphorylated receptor-associated Smad proteins (pR-Smad2 and pR-Smad3), whereas Smad4-positive lines did not. These elevated pR-Smad levels could not be attributed to a decreased dephosphorylation rate nor to increased expression of TGF-β type I (TβR-I) or type II (TβR-II) receptors. Although minimal amounts of free bioactive TGF-β1 and TGF-β2 were detected in conditioned medium, treatment with a pan-specific (but not a TGF-β3 specific) TGF-β-neutralizing antibody and with anti-αVβ6 integrin antibody decreased steady-state pSmad2 levels and activation of a TGF-β-inducible reporter gene in neighboring cells, respectively. Thus, activation of TGF-β at the cell surface was responsible for the increased autocrine endogenous and paracrine signaling. Blocking TβR-I activity using a selective kinase inhibitor (SD-093) strongly decreased the in vitro motility and invasiveness of the pancreatic carcinoma cells without affecting their growth characteristics, morphology, or the subcellular distribution of E-cadherin and F-actin. Moreover, exogenous TGF-β strongly stimulated in vitro invasiveness of BxPC-3 cells, an effect that could also be blocked by SD-093. Thus, the motile and invasive properties of Smad4-deficient pancreatic cancer cells are at least partly driven by activation of endogenous TGF-β signaling. Therefore, targeting the TβR-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.

Role of p38 mitogen activated protein kinase in a model of osteosarcoma-induced pain.

The focus of this work was to examine the potential role of p38 mitogen activated protein kinase (p38) in a mouse model of bone cancer (osteosarcoma) pain. To generate osteosarcoma and sham animals, osteosarcoma cells or medium were injected into the medullary canal of the femur. Initially, ipsilateral tactile allodynia was observed in both groups, but by 12 days post-surgery, thresholds in the sham group returned towards baseline while hypersensitivity in the osteosarcoma group lasted throughout the study. An increase in phosphorylated p38 was detected by western blotting in dorsal root ganglia (DRG) and spinal cord day 14 after surgery. Immunohistochemistry showed that p38 was phosphorylated in DRG and spinal dorsal horn neurons at this time point. Two doses of a selective p38 inhibitor, SCIO-469, were administered in the chow starting 5 days post-surgery and continued throughout the study. Treatment with SCIO-469 led to a decrease in osteosarcoma-induced clinical score but had no effect on the allodynia. Bone erosion and tumor growth were also examined but no significant reduction of bone erosion or tumor growth was observed in the SCIO-469 treated mice. These data suggest that the p38 signaling pathway does not play a major role in bone cancer-mediated pain.

Abstract 4515: SPR965: an oral PI3K/ mTOR C1/C2 inhibitor for the treatment of solid tumors

Background The discovery and development of inhibitors for the PI3K/AKT/mTOR signaling pathway is an attractive area of research due to its association with several oncogenic malignancies. This signaling pathway controls cellular growth as well as survival via regulation of widely divergent physiological processes, i.e. cell cycle progression, differentiation, transcription, translation and apoptosis. Constitutive activation of the PI3 kinase alpha and/or the downstream protein mTOR has been implicated in the progression of a large variety of solid tumors. Literature reports suggest the importance of developing combined and specific inhibitor of both PI3K and mTOR kinases. Described herein is the discovery of a novel small molecule, SPR965, a potent, and orally bioavailable inhibitor for class 1 PI3 Kinase and mTOR kinases with the potential for the clinical treatment of various solid tumors, specifically prostate, ovarian and colon cancers. The complete preclinical profile of SPR965 is presented in detail. Methods Drug candidates were evaluated in an in vitro enzyme assays to determine their inhibitory activity PI3 and mTOR C1/ C2 kinases. Promising candidates were then evaluated in proliferation assays using various human cancer cell lines (PC3 - prostate, SKOV3 - ovarian, HCT-116 - colon and A2780 - ovarian). The most promising leads were then evaluated against a panel of 456 kinases to determine the level of selectivity for our primary targets - PI3K and mTOR C1/C2. Compounds meeting the desired threshold of potency and selectivity were evaluated for their in vivo pharmacokinetic profile (iv/ po) in rodents followed by studies in mouse xenograft models (SKOV3 and HCT-116). Results SPR965 is a potent inhibitor of PI3K alpha and mTOR with an IC50 of 24 and 25 nM respectively. SPR965 is also a highly selective inhibitor of PI3 and mTOR C1/C2 kinases when evaluated in a screen against 456 kinases. Further studies demonstrated that SPR965 is a potent inhibitor of proliferation in a multiple cell lines and in several xenograft models. The proliferation inhibition activity (EC50) in A2780 is 17 nM, PC3 is 30 nM, SKOV3 is 74 nM, and HCT-116 is 163 nM. SPR965 is one of the most efficacious PI3/mTOR kinase inhibitor yet reported, with ED50 = 0.5 mg/Kg as determined in a SKOV3 xenograft mouse model and ED50 = 0.6 mg/ Kg in HCT-116 xenograft mouse model. This dose level is markedly lower than those reported for other reported inhibitors of this pathway. Pharmacokinetic studies in Sprague Dawley rats and in NUDE mice indicated that the oral bioavailability of SPR965was ∼100% and 75% respectively. Conclusion SPR965 is one of the most efficacious inhibitor of the PI3 and mTOR kinases when compared to the reported ED50s of other reported compounds. It also is one of the most selective and highly bioavailable inhibitor of the PI3 and mTOR kinases. Further studies are underway to support first-in-human trials with SPR965 where we hope to demonstrate its unique therapeutic benefits. Citation Format: Reena Arora, Bakul K. Dutta, Ravinder Goel, Frank P. Hollinger, Bilash Kulia, Dinesh Mahajan, Amal R. Mahapatra, Milind Sagar, Somdutta Sen, Amit Sharma, Sundeep Dugar. SPR965: an oral PI3K/ mTOR C1/C2 inhibitor for the treatment of solid tumors. [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 4515. doi:10.1158/1538-7445.AM2014-4515

Abstract 2653: Preclinical activity of dual PI3K/mTOR inhibitor SPR965 in multiple myeloma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Relapsed and refractory multiple myeloma (MM) remains a significant clinical challenge and drugs with new mechanisms of action to overcome resistance are needed. Constitutive activation of the PI3K/Akt/mTOR pathway in MM promotes tumorigenesis through propagation of the cell cycle, protein synthesis, and inhibition of apoptosis. Anti-MM effects of rapalogs are limited due to feedback activation of the PI3K/Akt signaling pathway. Agents capable of targeting PI3K and mTORC1 or both mTORC1/C2 are able to partially overcome such resistance mechanisms. However, the mTORC2 mediated increase in pAkt (Ser473) after PI3K/mTORC1 inhibition and the activation of PI3K after mTORC1/C2 inhibition could still contribute to resistance to such agents. We sought to investigate the effects of SPR965 (synthesized and provided by Sphaera Pharma, Singapore under an MTA), an orally bioavailable novel small molecule with inhibition of class 1 PI3 kinase and mTORC1/C2 kinases on MM cell lines and patient cells. Results: SPR965 induced cytotoxicity and inhibited proliferation in all MM cell lines examined with IC50 values between 25-500nM. To understand if SPR965 induced apoptotic cell death, annexin/PI staining followed by flow cytometric assays were performed, which showed a clear increase in cells undergoing apoptosis. Western blots showed increase in caspase-3, -9, and PARP cleavage confirming apoptotic induction by SPR965. Importantly, SPR965 caused potent increase in apoptosis in cytogenetically distinct MM patient cells. Next, we examined the mechanism of action of SPR965. SPR965 caused potent mTORC1 inhibition evidenced by decreased p4E-BP1, p-p70S6K, and pS6 at doses as low as 25nM. SPR965 was able to inhibit PI3K activity as shown by decreased pPDK1 and pBTK at slightly higher concentrations of 75nM. Phosphorylation of Akt S473, a TORC2 substrate, increased at initial low concentrations of SPR965, but was attenuated at concentrations of 100 nM and above, demonstrating dose-dependent inhibition of mTORC2. Such effects were also observed when we performed western blots on MM patient derived primary plasma cells. Increased levels of p27 (KIP1) were observed suggestive of G1 growth arrest, which was confirmed on cell cycle analysis. Since SPR965 caused an increase in pAkt (both T308 and S473) at doses up to 75nM, we examined if SPR965 was able to synergize with an Akt inhibitor MK2206 at doses lower than 75nM. Our results showed potent synergy suggesting that the pAkt up regulation contributes to partial resistance, which is inhibited by SPR965 at doses of 100nM and higher, doses that are still clinically achievable. Conclusion: Our findings demonstrate SPR965 induces apoptosis of MM cells through the inhibition of PI3K and mTORC1/C2 activity. Further studies are underway to better characterize the mechanism of action of SPR965, all of which will be informative for the drug to be used as a single agent or in combination with other agents in relapsed MM. Citation Format: Jeremy T. Larsen, Vijay Ramakrishnan, Jessica Haug, Teresa Kimlinger, Somdutta Sen, Dinesh Mahajan, Sundeep Dugar, S. Vincent Rajkumar, Shaji K. Kumar. Preclinical activity of dual PI3K/mTOR inhibitor SPR965 in multiple myeloma. [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 2653. doi:10.1158/1538-7445.AM2015-2653