Pomalidomide and lenalidomide induce p21 WAF-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 Burkitt's 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.

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.

SP500263, a novel SERM, blocks osteoclastogenesis in a human bone cell model: role of IL-6 and GM-CSF.

Bone metabolism requires tightly coupled activities exhibited by two unique cell populations, the bone-resorbing osteoclasts and the bone-forming osteoblasts. Imbalance in the function of these two cell types can result in osteoporosis, a condition characterized by loss in bone integrity and of bone mass. We developed a human bone cell culture model that allows the in vitro study of bone formation and osteoclastogenesis and employed this bone model for the screening and pharmacological analyses of protein and small molecule therapeutics. The cytokines, interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF), play an intricate role in osteoclastogenesis in this system. Neutralizing antibodies to IL-6 and GM-CSF decreased the formation of osteoclast-like cells. SP500263, an early lead compound from a novel class of selective estrogen receptor modulators (SERMs), was more efficacious than estrogen and comparable to raloxifene in blocking cytokine production and formation of osteoclast-like cells. Our research demonstrates the usefulness of the in vitro co-culture model in the dissection of molecular events relevant to bone metabolism and provides greater insight into a potential novel role for cytokines in bone resorption. Furthermore, representatives of the SP500263 family of SERMs may be effective as therapeutics for the treatment of osteoporosis.