Dominique Verhelle

Lenalidomide and CC-4047 Inhibit the Proliferation of Malignant B Cells while Expanding Normal CD34+ Progenitor Cells

Clinical studies involving patients with myelodysplastic syndromes or multiple myeloma have shown the efficacy of lenalidomide by reducing and often eliminating malignant cells while restoring the bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide and a structurally related analogue, CC-4047, on the proliferation of two different human hematopoietic cell models: the Namalwa cancer cell line and normal CD34+ progenitor cells. Both compounds had antiproliferative effects on Namalwa cells and pro-proliferative effects on CD34+ cells, whereas p21WAF-1 expression was up-regulated in both cell types. In Namalwa cells, the up-regulation of p21WAF-1 correlated well with the inhibition of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest, whereas in CD34+ progenitor cells the increase of p21WAF-1 did not inhibit proliferation. Similarly, antiproliferation results were observed in two B lymphoma cell lines (LP-1 and U266) but interestingly not in normal B cells where a protection of apoptosis was found. Finally, CC-4047 and lenalidomide had synergistic effects with valproic acid [a histone deacetylase (HDAC) inhibitor] by increasing the apoptosis of Namalwa cells and enhancing CD34+ cell expansion. Our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus normal cells and could explain, at least in part, the reduction of malignant cells and the restoration of bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, proposes new potential clinical uses, such as bone marrow regeneration, and suggests that the combination of lenalidomide or CC-4047 with certain HDAC inhibitors may elevate the therapeutic index in the treatment of hematologic malignancies.

Pomalidomide and Lenalidomide Induce p21WAF-1 Expression in Both Lymphoma and Multiple Myeloma through a LSD1-Mediated Epigenetic Mechanism

Lenalidomide and pomalidomide have both been evaluated clinically for their properties as anticancer agents, with lenalidomide being available commercially. We previously reported that both compounds cause cell cycle arrest in Burkitts lymphoma and multiple myeloma cell lines by increasing the level of p21(WAF-1) expression. In the present study, we unravel the molecular mechanism responsible for p21(WAF-1) up-regulation using Namalwa cells as a human lymphoma model. We show that the increase of p21(WAF-1) expression is regulated at the transcriptional level through a mechanism independent of p53. Using a combination of approaches, we show that several GC-rich binding transcription factors are involved in pomalidomide-mediated up-regulation of p21(WAF-1). Furthermore, we report that p21(WAF-1) up-regulation is associated with a switch from methylated to acetylated histone H3 on p21(WAF-1) promoter. Interestingly, lysine-specific demethylase-1 (LSD1) silencing reduced both pomalidomide and lenalidomide up-regulation of p21(WAF-1), suggesting that this histone demethylase is involved in the priming of the p21(WAF-1) promoter. Based on our findings, we propose a model in which pomalidomide and lenalidomide modify the chromatin structure of the p21(WAF-1) promoter through demethylation and acetylation of H3K9. This effect, mediated via LSD1, provides GC-rich binding transcription factors better access to DNA, followed by recruitment of RNA polymerase II and transcription activation. Taken together, our results provide new insights on the mechanism of action of pomalidomide and lenalidomide in the regulation of gene transcription, imply possible efficacy in p53 mutated and deleted cancer, and suggest new potential clinical uses as an epigenetic therapy.

Pomalidomide and lenalidomide regulate erythropoiesis and fetal hemoglobin production in human CD34 + cells

Sickle-cell disease (SCD) and beta thalassemia constitute worldwide public health problems. New therapies, including hydroxyurea, have attempted to augment the synthesis of fetal hemoglobin (HbF) and improve current treatment. Lenalidomide and pomalidomide are members of a class of immunomodulators used as anticancer agents. Because clinical trials have demonstrated that lenalidomide reduces or eliminates the need for transfusions in some patients with disrupted blood cell production, we investigated the effects of lenalidomide and pomalidomide on erythropoiesis and hemoglobin synthesis. We used an in vitro erythropoiesis model derived from human CD34+ progenitor cells from normal and SCD donors. We found that both compounds slowed erythroid maturation, increased proliferation of immature erythroid cells, and regulated hemoglobin transcription, resulting in potent induction of HbF without the cytotoxicity associated with other HbF inducers. When combined with hydroxyurea, pomalidomide and, to a lesser extent, lenalidomide were found to have synergistic effects on HbF upregulation. Our results elucidate what we believe to be a new mechanism of action of pomalidomide and lenalidomide and support the hypothesis that pomalidomide, used alone or in combination with hydroxyurea, may improve erythropoiesis and increase the ratio of fetal to adult hemoglobin. These findings support the evaluation of pomalidomide as an innovative new therapy for beta-hemoglobinopathies.

Immunomodulatory Drug CC-4047 is a Cell-type and Stimulus-Selective Transcriptional Inhibitor of Cyclooxygenase 2

COX2 (prostaglandin G/H synthase, PTGS2) is a well-validated target in the fields of both oncology and inflammation. Despite their significant toxicity profile, non-steroidal anti-inflammatory drugs (NSAIDs) have become standard of care in the treatment of many COX2-mediated inflammatory conditions. In this report, we show that one IMiDs® immunomodulatory drug, CC-4047, can reduce the levels of COX2 and the production of prostaglandins (PG) in human LPS-stimulated monocytes. The inhibition of COX2 by CC-4047 occurs at the level of gene transcription, by reducing the LPS-stimulated transcriptional activity at the COX2 gene. Because it is a transcriptional rather than an enzymatic inhibitor of COX2, CC-4047 inhibition of PG production is not susceptible to competition by exogenous arachadonic acid (AA). The distinct mechanisms of action allow CC-4047 and a COX2-selective NSAID to work additively to block PG secretion from monocytes. CC-4047 does not, however, block COX2 induction in or prostacyclin secretion from IL-1β stimulated human umbilical vein endothelial cells (HUVEC) cells, nor does it inhibit COX1 in either monocytes or HUVEC cells. CC-4047 also inhibits COX2 and PG production in monocytes derived from patients with sickle cell disease (SCD). Taken together, the data in this manuscript suggest CC-4047 will provide important anti-inflammatory benefit to patients and will improve the safety of NSAIDs in the treatment of SCD or other inflammatory conditions.

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

Abstract 3518: Growth inhibition of SCLC cell lines by treatment with LSD1 inhibitors is associated with modulation of neuroendocrine pathways

Small cell lung cancer (SCLC) is an aggressive malignancy with a high propensity for early metastasis. Response rates to first-line chemotherapy are typically high, but short lived. We describe an epigenetic-based mechanism for targeting SCLC using inhibitors for the LSD1 histone lysine demethylase. Mechanistically reversible and irreversible LSD1 inhibitors (GSK690 and OG-86) demonstrate induction of either cell cycle arrest or apoptosis in 54% of SCLC cell lines (12/22) that becomes apparent upon continuous treatment for 7-to-10 days. Maximal rates of growth inhibition in sensitive cell lines vary from 50-to-95% and plateaus between 18-to-21 days of LSD1 inhibitor treatment. Thus, heterogeneous sensitivity to LSD1 inhibitors exists between cell line models as well as within subpopulations of cells in the same cell line. To understand the mechanisms underlying LSD1 inhibitor activity in SCLC we have performed RNA-seq and ChIP-seq experiments coupled with bioinformatic analysis of expression signatures in sensitive and resistant models. Our data indicate that although LSD1 is over-expressed in SCLC cell lines and patient samples relative to non-small cell lung cancers, high LSD1 expression does not predict sensitivity to LSD1 inhibitors. Pathway analysis demonstrates that LSD1 inhibition modulates the expression of genes involved in cell adhesion and axon guidance including members of the Ephrin and Semaphorins families. At LSD1 target genes, we demonstrate site-specific H3K4me2 histone methylation changes overlapping LSD1 binding sites, however no global changes in H3K4me2 were observed. Interestingly we find morphological and cell adhesion changes in sensitive cell lines that coincide with expression changes in markers of neuroendocrine differentiation of SCLC such as GRP, NCAM, and NEUROD1. Based on these data, we propose a model that LSD1 inhibition modulates the neuroendocrine differentiation program of SCLC cells promoting tumor inhibition in sensitive SCLC models. Citation Format: Thomas A. Paul, Shikhar Sharma, Jill Hallin, Tao Xie, Timothy Nichols, Mike Greig, James Hardwick, Martin Wythes, Dominique Verhelle. Growth inhibition of SCLC cell lines by treatment with LSD1 inhibitors is associated with modulation of neuroendocrine pathways. [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 3518. doi:10.1158/1538-7445.AM2015-3518