Small molecule CKIa inhibitors as PI inducers and anti-cancer agents

A Recent study has implicated lenalidomide in the degradation of CKIα, as the drug mechanism of action in the pre-leukemia syndrome MDS with 5q deletion, indicating that CKIα may be a valid cancer drug target. CKIα ablation appears as a promising means of activating p53 and killing leukemia cells in MDS and AML; yet, with no selective CKIα inhibitors for in vivo use, the therapeutic value of CKIα inhibition cannot be validated. We found that CKIα ablation or degradation which activates p53 was not sufficient to selectively eliminate leukemia stem cells, due to robust expression of anti-apoptotic oncogenes driven by super-enhancer (SE) activation. We succeeded in developing CKIα inhibitors, targeting the catalytic pocket of CKIα and a special class which co-target CDK9 and suppress the RNA Pol II elongation factor P-TEFb (CDK9-CyclinT1 complex). We found that CKIα inhibitors possessing potent anti-leukemia activity are distinguished by their capacity to inhibit P-TEFb, evident by blocking the phosphorylation of RNA Pol II C-terminal domain (pCTD2/5), which controls the rate limiting step transcription and functions as a gatekeeper of SEs. The efficient P-TEFb-targeting activity of the inhibitors enables a selective blockade of leukemia SE-driven transcription, demonstrated by ChIP-seq analysis. As a result, SE-dependent transcription of major leukemia drivers including Myc and the anti-apoptotic oncogenes Bcl-2 and Mcl-1 was nearly abolished. Dramatic therapeutic effect of co-targeting CKIα and P-TEFb in leukemia, whereby combination of rapid p53 induction with disruption of oncogenic super-enhancers eliminates leukemia stem cells without compromising normal HSPCs. Indeed, at the therapeutic dose, we did not observe any significant adverse treatment effects even at daily treatment for 45 days in mice and explain the powerful and specific anti-leukemic therapeutic effects of this new class of inhibitors in-vivo (Minzel, Venkatachalam et al, Cell, under revision). These dual CKIα-CDK9 inhibitors are being evaluated now for clinical use in leukemia therapy. We are now establishing a few mouse models for testing these inhibitors in several types of cancers such as Melanoma and Colorectal Cancer. We have already established a mouse model of colorectal cancer based on APC deletion via CDX2-Cre-ER. The outcome of all these models will determine the feasibility of using the CKI inhibitors for the treatment of cancer types other than leukemia.

Gut pathophysiology due to expression of mutant p53 in a mouse model

The TP53 gene is the most important tumor suppressor in the cell. p53 is also the most mutated protein in human cancers, with more than 50% of tumors exhibiting a mutation at this locus. The vast majority of cancer-associated mutations in TP53 are missense mutations, resulting in the translation of an erroneous amino acid in the full-length protein. When mutated, p53 not only loses its tumor suppressive activity, but also gains new tumor-promotive features. p53 mutation is frequent in colorectal cancer, and is thought to mediate the transition from a benign polyp into an invasive carcinoma. Upon deletion of both CKIα and p53 (CKIα/p53Δgut), intestinal homeostasis is disrupted and mice develop invasive tumors. By breeding the CKIαfl/fl mice with mice carrying the p53 mutant allele R172H (corresponding to the human hot-spot mutation R175H), we were able to examine and compare the tissue effects of WT and mutant p53. We found that compared to CKIαΔgut mice, which showed robust activation of WT p53, p53 target-genes expression was completely absent in the homozygous mutant mice (CKIαΔgut/p53R172H/R172H). Surprisingly, CKIαΔgut/p53R172H/R172H mice had less carcinomas and lower proliferation rate in the small bowel, compared to CKIα/p53Δgut mice. Further investigation showed this suppression is mediated by epigenetic effects and is controlled by the gut microbiota. We are further studying the pathophysiological significance and mechanistic aspects of this surprising observation.

Studying low-grade atypical inflammation that promotes cancer

Inflammation is emerging as one of the hallmarks of cancer, yet we still do not understand its role in most tumors. Whereas a minority of solid tumors is associated with overt inflammation, long-term treatment with non-steroidal anti-inflammatory drugs (NSAIDs) is remarkably effective in reducing both intestinal tumorigenesis in a mouse model and mortality rates of major human solid tumors, by up to 75 percent. These figures indicate that inflammation may have many unrecognized faces, of which an indolent course may be far more important factor in cancer than previously appreciated. Medzhitov (2008) has coined a low-grade inflammatory response parainflammation (PI), referring to an adaptive response due to tissue stress or malfunction. Based on our studies (Elyada et al, Nature 2011, Pribluda et al, Cancer Cell 2013), we presume that this theoretical concept may apply to cancer, now with experimental evidence. Whereas classical inflammatory reactions are primarily associated with cell-exogenous insults, parainflammation appears to originate mostly upon cell-endogenous insults, such as DNA damage, or the DNA damage response. Using mouse models, we found that one of the most effective means of inducing PI is deleting or downregulating CKα in the epithelium. PI can then affect cancer in two opposing directions: while p53 is WT, PI suppresses carcinogenesis; on the other hand, in the absence of functional p53, PI promotes carcinogenesis (Pribluda et al, Cancer Cell, 2013). Parainflammation can be identified from a 40-gene signature and is found in both carcinoma cell lines and a variety of primary tumors, independently of tumor microenvironment (Aran, Lasry et al, Genome Biol 2016). We suggest that parainflammation may serve as a novel biomarker for screening patients who may particularly benefit from NSAID treatment.

Alumni

Dr. Audrey Lasry, Postdoctoral Fellow, Julie Blander’s lab, Weill Cornell Medical College, NY, audreylasry@gmail.com

Dr. Waleed Minzel, Postdoctoral Associate, Jonathan Kipnis Lab. The University of Virginia. waleedminzel@gmail.com 

Dr. Guy Brachya, Staff scientist /Researcher., Gamida Cell Ltd., Jerusalem, ISR, guy.brachya@mail.huji.ac.il

Dr. Ariel Pribluada, Postdoctoral Fellow., Genentech, pribluda@gmail.com

Dr. Ela Elyada, Postdoctoral Fellow at David Tuveson Lab, Cold Spring Harbor Laboratory (CSHL), Cold Spring Harbor, NY., ela.elyada@mail.huji.ac.il

Dr. Moshe Biton, Postdoctoral Fellow at Aviv Regev Lab, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts., bitonm@gmail.com

Dr. Elad Horwitz, Postdoctoral Fellow, Whitehead Institute for Biomedical Research, MIT.,  elad.horwitz@mail.huji.ac.il

Dr. Naama Kanarek, Postdoctoral Fellow at the Sabatini Lab Whitehead Institute for Biomedical Research, MIT., kanarekn@gmail.com

Dr. Robert Goldstein., Independent consultant in biology & pharma development, Innovo Mimetics Ltd., robert.e.goldstein@gmail.com

Dr. Marganit Farago-tirosh, Lab Manager at Prof. Yehudit Bergman lab, School of Medicine-IMRIC-Developmental Biology and Cancer Research., marganitf@ekmd.huji.ac.il

Dr. Shiri Shkedi-Rafid, director for the genetic counselling master program at the Hebrew University of Jerusalem, and a principal genetic counsellor at the Hadassah Medical Centre, Jerusalem ., shkedia@hadassah.org.il

Dr. Sharon Amit M.D., Director, Clinical Microbiology Unit, Hadassah Medical Center.,  sharonamit@hadassah.org.il

Prof. Yaron Avraham, The Jack & Simon Djanogly Professorial Chair in Biochemistry, Weizmann Institute of Science., avraham.yaron@weizmann.ac.il

Dr. Matti Davis, Ph.D. Analyst at Israel Biotech Fund, Israel Biotech Fund., davis.matti@gmail.com

Dr. Iris Lavon, Head, Molecular Neuro-Oncology Laboratory, Gaffin Center for Neuro-oncology, Hadassah Medical Center.,   irisl@hadassah.org.il

Prof. Steffen Jung, Head, Department of Immunology, Weizmann Institute of Science, s.jung@weizmann.ac.il

Prof Yardena Samuels, Department of Molecular Cell Biology, Weizmann Institute of Science, yardena.samuels@weizmann.ac.il

Dr. Ayelet Avraham, Lab head, Oncology Research Laboratory, Assaf Harofeh Medical Center.,  ayeleta@asaf.health.gov.il

Prof. Offer Gerlitz, Senior Staff lecturer, Developmental Biology and Cancer Research, School of Medicine IMRIC, the Hebrew University., offerg@ekmd.huji.ac.il

Prof. Michal Zion., Associate Professor/Member of the M.A. Committee and Coordinator of the Science Education Program and the Biology Teaching Program, Bar-Ilan University., michal.zion@biu.ac.il

Dr. Myriam Golembo, Staff scientist /Researcher., Protalix Biotherapeutics, myriam.golembo@gmail.com

Dr. Asne Bauskin., Centre for Immunology, St. Vincent’s Hospital and University of New South Wales, Sydney, Australia

Research projects

• Elucidating the role of autophagy in controlling parainflammation
• Studying p53 mutants in intestinal cancer
• Developing small molecule CKIa inhibitors as anti-cancer agents

1. W. Minzel, A. Venkatachalam, A. Fink, E. Hung, G. Brachya, I. Burstain, M. Shaham, A. Rivlin, I. Omer, A. Zinger, S.
   Elias, E. Winter, P.E. Erdman, R.W. Sullivan, L. Fung, F. Mercurio, D. Li, J. Vacca, N. Kaushansky, L. Shlush, M. Oren, R. Levine, E. Pikarsky, I. Snir-Alkalay, Y. Ben-Neriah Small molecules co-targeting ckialpha and the transcriptional kinases CDK7/9 control AML in preclinical models Cell, 175 (2018), pp. 171-185 e25
2. Morgenstern Y, Adhikari UD, Ayyash M, Elyada E, Tóth B, Moor A, Itzkovitz S, Ben‐Neriah Y. Casein kinase 1‐epsilon or 1‐delta required for Wnt‐mediated intestinal stem cell maintenance. The EMBO journal. 2017 Sep 28:e201696253.
3. Chang CH, Kuo CJ, Ito T, Su YY, Jiang ST, Chiu MH, Lin YH, Nist A, Mernberger M, Stiewe T, Ito S, Wakamatsu K, Alkalay, I and Ben-Neriah Y. CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation. Proceedings of the National Academy of Sciences. 2017 Sep 6:201702763.
4. Lasry A, Aran D, Butte AJ, Ben-Neriah Y. Cancer Cell–Autonomous Parainflammation Mimics Immune Cell Infiltration. Cancer research. 2017 Jul 15;77(14):3740-4.
5. Aran D, Lasry A, Zinger A, Biton M, Pikarsky E, Hellman A, Butte AJ, Ben-Neriah Y. Widespread parainflammation in human cancer. Genome biology. 2016 Dec;17(1):145.
6. Lasry A, Zinger A, Ben-Neriah Y. Inflammatory networks underlying colorectal cancer. Nat Immunol. 2016 Mar;17(3):230-40.
7. Lasry A, Ben-Neriah Y. Senescence-associated inflammatory responses: aging and cancer perspectives. Trends Immunol. 2015 Apr;36(4):217-28.
8. Kanarek N, Grivennikov SI, Leshets M, Lasry A, Alkalay I, Horwitz E, Shaul YD, Stachler M, Voronov E, Apte RN, Pagano M, Pikarsky E, Karin M, Ghosh S, Ben-Neriah Y. Critical role for IL-1β in DNA damage-induced mucositis. Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):E702-11.
9. Horwitz E, Stein I, Ben-Neriah Y, Pikarsky E. Animal model studies indicate a candidate biomarker for sorafenib treatment of hepatocellular carcinoma. Mol Cell Oncol. 2014 Oct 30;2(1):e968028.
10. Pribluda A, Elyada E, Wiener Z, Hamza H, Goldstein RE, Biton M, Burstain I, Morgenstern Y, Brachya G, Billauer H, Biton S, Snir-Alkalay I, Vucic D, Schlereth K, Mernberger M, Stiewe T, Oren M, Alitalo K, Pikarsky E, and Ben-Neriah Y. A senescence-inflammatory switch from cancer-inhibitory to cancer-promoting mechanism. Cancer cell. 2013 Aug 12;24(2):242-56.
11. Elyada E, Pribluda A, Goldstein RE, Morgenstern Y, Brachya G, Cojocaru G, Snir-Alkalay I, Burstain I, Haffner-Krausz R, Jung S, Wiener Z, Alitalo K, Oren M, Pikarsky E, and Ben-Neriah Y. CKIα ablation highlights a critical role for p53 in invasiveness control. Nature. 2011 Feb;470(7334):409.

• ERC, ISF Center of Excellence, AMRF, ICRF Profesorship