Marion Dorsch

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.

ALDH2(E487K) mutation increases protein turnover and promotes murine hepatocarcinogenesis.

Significance About 40% of East Asians and over 500 million people worldwide carry a specific polymorphism, ALDH2*2, and exhibit “Asian flush” after alcohol drinking. We generated a mouse strain with this engineered polymorphism and demonstrated its resemblance to human carriers in terms of defective alcohol metabolism. With this model, we show that murine ALDH2*2 increases ALDH2 protein turnover and promotes chemical-induced liver tumor development. Importantly, ALDH2 is unstable in ALDH2*2 human liver samples and is significantly down-regulated in human liver tumors. Data from our mouse and clinical studies suggest that ALDH2 is a liver tumor suppressor and the ALDH2*2 polymorphism is a risk factor for liver cancer. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele–alcohol interaction may be an even greater human public health hazard than previously appreciated.

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

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnPRP4 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.nnCitation 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 1135: Chemical Proteomics effort identifies PKN1 as a key player in the canonical NF-κB signaling pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnDeregulation of NF-κB signaling pathway has been identified as one of the key drivers of many hematological malignancies. Activation of the NF-κB pathway in these cells can happen either through activating mutations within the pathway, loss of function mutations of inhibitory molecules or through interaction with the stroma that secrete specific cytokines. The strategies for blocking the NF-κB pathway activation have so far focused on NF-κB pathway kinase inhibitors or the more general proteasome inhibitors. However, the adverse events associated with available NF-κB pathway modulators in pre-clinical and clinical settings have limited their general therapeutic use. To discover new targets and modulators of this key survival pathway, we performed a cellular phenotypic screen that led to the identification of a novel class of small molecules that inhibit the activation of the canonical NF-κB pathway. The compounds showed potent inhibition of NF-κB activation by multiple receptors including the TNF, antigen and BAFF/ APRIL receptors. Structure activity relationship analysis for this novel class of molecules identified pairs of stereoisomers wherein the cis isoform showed greater than 100 fold activity than the trans isoform. Detailed interrogation of the mechanism of action of the active cis isomers using biochemical and cellular pathway analysis revealed that the compounds are potent inhibitors of RHO associated kinases, only weakly inhibit IKK1 and IKK2 and strongly inhibit the induction of phosphorylated IkBα upon stimulation. However, the differential activity of the stereoisomers was not seen for the inhibition of the RHO associated kinases, suggesting the inhibition of NF-κB pathway was due to modulation of other targets. This raises the possibility of identifying novel targets associated with this compound. Using in-lysate affinity chemical proteomics with the active and in-active enantiomer, we have identified PKN1 as a key efficacy target for the inhibitors. Knockdown of PKN1 using genetic tools was sufficient to abrogate the response of cells to TNFα stimulation implicating a critical role for PKN1 in the activation of NF-κB pathway. Our findings therefore present an opportunity to develop novel NF-κB inhibitors for the tumors that depend on this key pathway for survival.nnCitation Format: Katherine Tang, Michael Lampa, Tieu-Binh Le, Matthieu Barrague, Ronald Tomlinson, Brittain Scott, Tahir Majid, Marion Dorsch, Hong Cheng, Christoph Lengauer, Carlos Garcia-Echeverria, Francisco Adrian, Mikhail Levit, Balin Zhang, Kin Yu, Ivan Cornella-Taracido, Lakshmi Srinivasan. Chemical Proteomics effort identifies PKN1 as a key player in the canonical NF-κB signaling pathway. [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 1135. doi:10.1158/1538-7445.AM2013-1135

Abstract 1796: SAR302503: A Jak2 inhibitor with antitumor activity in solid tumor models

Numerous studies have recognized the critical role of STAT3 in malignant transformation and tumor progression. Constitutive STAT3 activation is frequently found in cancer cell lines and tumor samples and it is usually linked to the presence of IL-6. Autocrine or paracrine IL-6 loops have been described to provide tumor cells with the ability to proliferate, survive, migrate and metastasize. At the molecular level, IL-6 binding to its receptor results in activation of Jak/STAT3 signaling and other signaling cascades, namely PI3K/Akt, MEK/ERK1-2, with a well established role in cancer. We have evaluated the ability of SAR302503, a selective Jak2 inhibitor entering a PhIII clinical trial in myelofibrosis patients, to block these pathways in a panel of ∼20 tumor cell lines representing different cancer types (prostate, breast, lung, colorectal, pancreas, hepatocellular, etc). A 45 minutes treatment with different concentrations of SAR302503 (0.1-10 µM) was sufficient to block both basal or IL-6 induced STAT3 phosphorylation in a dose dependent manner. Compound concentrations equal or greater than 0.1 µM were able to reduce the phosphorylation levels of STAT3 by an extent greater than 50% in all the cell lines included in the study. The impact of SAR302503 in tumor cell proliferation and survival was evaluated using different assays including clonogenic assays. Complete inhibition of colony formation was achieved at concentrations of SAR302503 below 1 µM in most of the cell lines. The antitumor activity of SAR302503 was evaluated in mice xenotransplanted subcutaneously with DU145 human prostate cancer cells. Oral administration of SAR302503 for 10 days resulted in significant dose dependent tumor growth inhibition, near to stasis at the highest dose (T/C=19% at 100 mg/kg, bid). In summary, we demonstrate that SAR302503 negatively impacts the proliferation and survival of different solid tumor cells and our data supports a role for a selective Jak2 inhibition in treating solid tumors with activated Jak/STAT signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1796. doi:1538-7445.AM2012-1796