Anlai Wang

Interfering with Resistance to Smoothened Antagonists by Inhibition of the PI3K Pathway in Medulloblastoma

Resistance of medulloblastoma to Smo antagonists can be delayed or prevented by specific drug combinations. An End Run Against Tumor Resistance Cancer cells are as clever as microbes. Mustering their considerable abilities to rapidly replicate and evolve, both cancer cells and bacteria quickly develop resistance to the drugs we use to fight them. Modern medicine confronts a growing population of pathogens that cannot be treated by our usual antibiotics, and oncologists must be prepared with second- and third-line therapies, because tumors that retreat from initial drug treatments often return with renewed vigor. Buonamici et al. confront this problem in their study of a new class of cancer therapeutic agents now in clinical trials—antagonists of a membrane protein called Smoothened (Smo). The Smo receptor normally regulates a developmental pathway but is abnormally activated in medulloblastoma (a malignant brain tumor) and basal cell carcinoma of the skin. Medulloblastomas in mice respond well to these Smo antagonists but soon become resistant, these authors find. If, however, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is added to the initial drug cocktail, resistance is delayed or even prevented. In some cancers, the Smo receptor is active even when its ligand is absent, conferring dependence of the tumor on the downstream Hedgehog signaling pathway, which ultimately regulates gene expression through the Gli transcription factors. Treatment of Smo-addicted tumors in mice with Smo antagonists ultimately leads to development of resistance, although tumor growth is inhibited for a while. The authors found that the tumors eluded the drug in several ways: The genes for the Gli transcription factors were sometimes amplified, compensating for loss of pathway stimulation. In other resistant tumors, there were point mutations in the Smo receptor itself that allowed reactivation of the pathway. In yet another group of tumors, by examining which genes were up-regulated, the authors found activation of a completely different signaling pathway—the PI3K pathway. Further experiments in medulloblastoma-bearing mice revealed that resistance could be delayed or even prevented by including a PI3K inhibitor along with the Smo antagonist in the initial treatment that tumor-bearing animals received. The PI3K inhibitor alone had no effect. By looking at resistance mechanisms to Smo antagonists before the drug is used in the clinic, the results of this study will better arm oncologists against the molecular defenses that cancers may commandeer to evade this drug. And by identifying a drug combination that delays or even combats development of resistance when used as a first-line treatment in clinical trials, these results could ultimately improve the lives of patients with medulloblastoma or other cancers that depend on Smo for their survival. The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.

Discovery of NVP-LEQ506, a Second-Generation Inhibitor of Smoothened

Inhibition of the Hedgehog (Hh) pathway targeting the Smoothened receptor has proven therapeutic benefit for the treatment of Hh-dependent cancers. Lead optimization provided a novel type of Smoothened inhibitor based on a pyridazine core resulting in the clinical compound NVP-LEQ506. This new agent combines high intrinsic potency and good pharmacokinetic properties resulting in excellent efficacy in preclinical rodent tumor models of medulloblastoma. Activity against a Smo mutant conferring resistance observed in a clinical trial with a competitor compound suggests additional therapeutic potential.

PIM inhibitors target CD25-positive AML cells through concomitant suppression of STAT5 activation and degradation of MYC oncogene

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.

Evaluation of cancer dependence and druggability of PRP4 kinase using cellular, biochemical, and structural approaches

Background: Little is known about the cancer dependence and druggability of PRP4. Results: Significance of PRP4 catalytic activity is demonstrated, novel substrates are identified, and features of kinase domain structure are revealed. Conclusion: PRP4 is required for cancer cell survival, displays substrate specificity, and is amenable to pharmacological inhibition. Significance: Our results indicate that PRP4 is a potential drug target to pursue in cancer. PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.

Abstract B26: Immunomodulatory activity of Isatuximab

Isatuximab, previously known as SAR650984 is a naked chimeric IgG1 anti-CD38 monoclonal antibody. It is currently in clinical trials demonstrating single-agent activity in heavily pretreated relapsed/refractory multiple myeloma (RRMM). Isatuximab induces killing of CD38 positive tumor cells by triggering Fc dependent effector functions such as ADCC, ADCP and CDC, as well as Fc independent direct apoptotic effect. However, its effects in immune cells that express CD38 are emerging and are not yet well defined. The aim of this study was to investigate the effects of Isatuximab on immune effector cells, to better understand its anti-tumor mechanisms underlying the clinical benefit. Among immune sub-populations, Natural Killer (NK) cells and monocytes have the highest CD38 receptor density. Further, NK cells and monocytes play critical roles in immunity against cancer. We investigated Isatuximab9s effect on these immune effector cells including their expansion, cytokine secretion and activation. Treatment (8 days at 1µg/ml) of 106 whole human peripheral blood mononuclear cells with Isatuximab induced expansion of NK and monocytes. In addition to enhance expansion, Isatuximab was able to induce the release of IFN-γ (~4000-7000 pg/ml) and TNF-α (~150-200 pg/ml) in NK cells in the absence of target cells. Incubation of Isatuximab (1µg/ml) for an hour also enhanced cytotoxic activity of human NK cells, both NK-92 cell line and isolated human NK cells, against CD38 negative K562 and JHH6 tumor cell lines. In the case of monocytes, Isatuximab activates phagocytosis by human monocytic cell line THP-1 and purified human monocytes. Furthermore, Isatuximab-activated NK cells can induce monocyte to M1 macrophage polarization through IFN-γ release in vitro. These findings collectively show evidence of Isatuximab being an immune-modulatory agent able to induce NK cell and monocyte activation. Further study is needed to determine whether Isatuximab9s immunomodulatory activity could be utilized for the treatment of both CD38-positive and negative cancers by enhancing its anti-tumor function. Citation Format: Srimathi S. Srinivasan, Anlai Wang, Zhili Song, Francisco Adrian. Immunomodulatory activity of Isatuximab. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B26.

Discovery of N-substituted 7-azaindoles as Pan-PIM kinase inhibitors – Lead series identification – Part II

N-Substituted azaindoles have been discovered as pan-PIM kinase inhibitors. Initial SAR, early ADME and PK/PD data of a series of compounds is described and led to the identification of promising pan-PIM inhibitors which validated our interest in the 7-azaindole scaffold and led us to pursue the identification of a clinical candidate.

Abstract 4376: Evaluation of PRP4 kinase as a potential drug target in cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC PRP4 kinase plays a crucial role in regulating pre-mRNA splicing, cell cycle progression, proliferation, and survival. The aforementioned functions have been well documented in Schizosaccharomyces pombe, Caenorhabditis elegans, and human cancer cell lines. For example, it was demonstrated that PRP4 kinase is essential for growth in fission yeast, and disruption of C. elegans PRP4 by RNAi resulted in a highly penetrant early embryonic lethality. In experiments utilizing siRNAs to screen for kinases essential for pancreatic cancer cell survival, PRP4 knockdown was demonstrated to increase apoptosis and decrease viability. In a genome-wide pooled shRNA screen, shRNAs against PRP4 was shown to reduce the viability of DLD-1, HCT-116, and HCC1954 cancer cell lines. Similarly, PRP4 kinase was identified as a potential therapeutic target in a pooled shRNA screen designed to identify genes required for proliferation and survival of diffuse large B-cell lymphoma cell lines. Moreover, in an effort to reveal potential kinase targets to treat multidrug resistance ovarian cancer, inhibition of PRP4 activity by shRNAs was shown to re-sensitize chemo-resistant human ovarian cancer to paclitaxel treatment. Interestingly, loss of PRP4 kinase was also demonstrated to enhance paclitaxel activity in breast cancer cells. To further investigate PRP4 kinase substrate spectrum and explore the druggability of PRP4 kinase, we utilize quantitative proteomics and structural biology to help achieve these objectives. In this report, we provided evidence that the kinase domain of PRP4 is essential for regulating cell growth and survival. In addition, through a global proteomics approach, we expanded the interactome and phosphoproteome of PRP4 kinase in cancer cells and identifed novel substrates of PRP4, including oncogenic PAK4 kinase. Subsequently, these substrates were substantiated in orthogonal biochemical and cellular assays. These new biological findings not only identify suitable biomarkers to monitor PRP4 kinase activity, but also provide interesting avenues for future in-depth interrogation of PRP4 functions in cancer biology and clinical development. Finally, we solve the X-ray structure of the PRP4 kinase domain and identify several features suitable for the rational design of PRP4 kinase inhibitors. We further provided the co-crystal structure of PRP4 kinase domain in complex with a small molecule and elucidated the exploitable mechanisms to synthesize potent and specific PRP4 inhibitors. Future efforts will be focused on understanding patient stratification strategy and assessing the utility of PRP4 kinase inhibitors in relevant pre-clinical models of cancer. Citation Format: Qiang Gao, Ingrid Mechin, Nayantara Kothari, Zhuyan Guo, Gejing Deng, Anlai Wang, Dmitri Wiederschain, Jennifer Rocnik, Werngard Czechtizky, Feng Liu, Tahir Majid, Vinod Patel, Christoph Lengauer, Carlos Garcia-Echeverria, Bailin Zhang, Hong Cheng, Marion Dorsch, Shih-Min A. Huang. Evaluation of PRP4 kinase as a potential drug target in cancer. [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 4376. doi:10.1158/1538-7445.AM2013-4376

Abstract 1976: The discovery of mechanisms of resistance to SMO antagonists and the therapeutic implications

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Smoothened (Smo) is a G-protein coupled receptor (GPCR)-like molecule that activates the Hedgehog (Hh) signal transduction pathway. In the resting state, the 12-pass transmembrane protein Patched (Ptch) inhibits Smo activity. When Ptch inhibition is attenuated, Smo signals via a cytosolic complex of proteins leading to activation of the Gli family of transcription factors. Genetic activation of the Hh pathway at or upstream of Smo is linked to tumorigenesis in several cancers. In particular, somatic mutations in Ptch and Smo leading to constitutive pathway activation are found in sporadic medulloblastoma (MB) and basal cell carcinoma (BCC). Evidence suggests that antagonists of Smo may abrogate the tumorigenic phenotype engendered by Ptch inactivation. NVP-LDE225 is a potent and selective orally available Smo antagonist that robustly inhibits Smo-dependent signaling in vitro and in vivo. NVP-LDE225 exerted dose-related anti-tumor activity in vivo in several genetically defined MB models that are driven by mutations in Ptch leading to near complete tumor regression and Hh pathway inhibition. However, following long-term continuous dosing of NVP-LDE225 in medulloblastoma allograft models, evidence of resistance to NVP-LDE225 was observed. Here, we describe our efforts to proactively identify mechanisms of resistance to targeted therapy of Smo. Genome-wide DNA- and RNA-profiling of resistant tumors revealed distinct resistance mechanisms allowing tumors to evade the inhibitory effects of Smo antagonists. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, was identified as one mechanism leading to restoration of pathway signaling despite adequate drug exposure. Additional mining of the gene expression data for pathway signatures that are selectively deregulated in resistant tumors identified increased phosphatidylinositol-3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we showed that the combination of NVP-LDE225 with the dual PI3K/mTor inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma. 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 1976.