Kyle Chan

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.

Cathepsin K Is a Critical Protease in Synovial Fibroblast-Mediated Collagen Degradation

Synovial fibroblasts (SFs) play a critical role in the pathogenesis of rheumatoid arthritis (RA) and are directly involved in joint destruction. Both SF-resident matrix metalloproteases and cathepsins have been implicated in cartilage degradation although their identities and individual contributions remain unclear. The aims of this study were to investigate the expression of cathepsin K in SFs, the correlation between cathepsin K expression and disease severity, and the contribution of cathepsin K to fibroblast-mediated collagen degradation. Immunostaining of joint specimens of 21 patients revealed high expression of cathepsin K in SFs in the synovial lining and the stroma of synovial villi, and to a lesser extent in CD68-positive cells of the synovial lining. Cathepsin K-positive SFs were consistently observed at sites of cartilage and bone degradation. Expression levels of cathepsin K in the sublining and vascularized areas of inflamed synovia showed a highly significant negative correlation with results derived from the Hannover Functional Capacity Questionnaire (r = 0.78, P = 0.003; and r = 0.70, P = 0.012, respectively) as a measure of the severity of RA in individual patients. For comparison, there was no correlation between Hannover Functional Capacity Questionnaire and cathepsin S whose expression is limited to CD-68-positive macrophage-like synoviocytes. The expression of cathepsin K was also demonstrated in primary cell cultures of RA-SFs. Co-cultures of SFs on cartilage disks revealed the ability of fibroblast-like cells to phagocytose collagen fibrils whose intralysosomal hydrolysis was prevented in the presence of a potent cathepsin K inhibitor but not by an inhibitor effective against cathepsins L, B, and S. The selective and critical role of cathepsin K in articular cartilage and subchondral bone erosion was further corroborated by the finding that cathepsin K has a potent aggrecan-degrading activity and that cathepsin K-generated aggrecan cleavage products specifically potentiate the collagenolytic activity of cathepsin K toward type I and II collagens. This study demonstrates for the first time a critical role of cathepsin K in cartilage degradation by SFs in RA that is comparable to its well-known activity in osteoclasts.

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.

The development of novel targeted therapeutics for treatment of multiple myeloma research roundtable

Participants: Kenneth C. Anderson, MD; Bart Barlogie, MD, PhD; James R. Berenson, MD; William S. Dalton, MD, PhD; Julian Adams, MD, PhD; Melissa Alsina, MD; Pirow Bekker, MD, PhD; Peter L. Bergsagel, MD; Lawrence Boise, PhD; Kyle Chan, PhD; Asher A. Chanan-Khan, MD; Raymond Comenzo, MD; Deborah Congdon; Peter Croucher, PhD; Joshua Epstein, MD, DSc; Robert Fenton, MD, PhD; Rafael Fonseca, MD; Gilles Gallant, PhD; Mohamad A. Hussein, MD; Sundar Jagannath, MD; Roger N. Pearse, MD, PhD; David G. Roodman, MD, PhD; Edward Sausville, MD, PhD; John D. Shaughnessy Jr., PhD; Keith Stewart, MD; David I. Stirling, PhD; Suzanne Trudel, MD; Brian Van Ness, MD; Thomas E. Witzig, MD

A novel fibroblast activation inhibitor attenuates left ventricular remodeling and preserves cardiac function in heart failure

Cardiac fibroblasts are critical mediators of fibrotic remodeling in the failing heart and transform into myofibroblasts in the presence of profibrotic factors such as transforming growth factor-β. Myocardial fibrosis worsens cardiac function, accelerating the progression to decompensated heart failure (HF). We investigated the effects of a novel inhibitor (NM922; NovoMedix, San Diego, CA) of the conversion of normal fibroblasts to the myofibroblast phenotype in the setting of pressure overload-induced HF. NM922 inhibited fibroblast-to-myofibroblast transformation in vitro via a reduction of activation of the focal adhesion kinase-Akt-p70S6 kinase and STAT3/4E-binding protein 1 pathways as well as via induction of cyclooxygenase-2. NM922 preserved left ventricular ejection fraction ( P < 0.05 vs. vehicle) and significantly attenuated transverse aortic constriction-induced LV dilation and hypertrophy ( P < 0.05 compared with vehicle). NM922 significantly ( P < 0.05) inhibited fibroblast activation, as evidenced by reduced myofibroblast counts per square millimeter of tissue area. Picrosirius red staining demonstrated that NM922 reduced ( P < 0.05) interstitial fibrosis compared with mice that received vehicle. Similarly, NM922 hearts had lower mRNA levels ( P < 0.05) of collagen types I and III, lysyl oxidase, and TNF-α at 16 wk after transverse aortic constriction. Treatment with NM922 after the onset of cardiac hypertrophy and HF resulted in attenuated myocardial collagen formation and adverse remodeling with preservation of left ventricular ejection fraction. Future studies are aimed at further elucidation of the molecular and cellular mechanisms by which this novel antifibrotic agent protects the failing heart. NEW & NOTEWORTHY Our data demonstrated that a novel antifibrotic agent, NM922, blocks the activation of fibroblasts, reduces the formation of cardiac fibrosis, and preserves cardiac function in a murine model of heart failure with reduced ejection fraction.

Abstract 4527: Oral multi-pathway inhibitors for the treatment of triple negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Triple negative breast cancer (TNBC: estrogen and progesterone receptor and Her2/Neu negative) is a high grade malignancy that is more aggressive with a higher rate of drug resistance, recurrence, metastasis, and death than other breast cancer subtypes. No targeted therapies exist, and women with TNBC have limited therapeutic options. There is clearly a need for new, more effective therapies for TNBC. We have previously demonstrated that two different classes of translation initiation inhibitors (represented by NM043 and NM922) are safe and effective in animal models of TNBC. Recent studies show that both classes of compounds have activities in the mTORC1 and mTORC2 pathways. In particular, NM043 inhibited phosphorylation of p70 S6 kinase at Thr389, phosphorylation of 4E-BP1 at Ser65, phosphorylation of Akt at Ser473, and phosphorylation of PI3 kinase at Tyr458. Phosphorylation of all of these proteins was similarly inhibited by NM922 but at higher concentrations than for NM043. Phosphorylation of PTEN was inhibited by NM043 but not NM922. Current research is aimed at identifying the tyrosine kinase-linked receptor pathway(s) and/or target(s) affected by NM043 and NM922 that result in inhibition of phosphorylation of PI3K. In addition to being effective in animal models of TNBC, NM922 (but not NM043) was shown to retain its anti-tumor effect after treatment withdrawal. Further, NM922 (but not NM043) was shown to prevent the TGF-β dependent activation of fibroblasts. These data indicate that in addition to anti-proliferative effects on the tumor itself, NM922 may act on the tumor stroma that supports the growth and development of the tumor, thus potentially decreasing recurrence and metastasis. Although there are several possible pathways by which TGF-β promotes fibroblast differentiation (including PI3K), the most likely additional pathway that NM922 affects is the Smad pathway. NM922 has good oral bioavailability and is at least as effective at treating TNBC when administered orally as when administered i.p. This, combined with its dual activities on both the primary tumor and the tumor stroma makes it a very attractive clinical candidate for reducing recurrence and metastasis and increasing survival rates in TNBC. NM922 has a novel mechanism of action and should be valuable both as a stand-alone therapy and in conjunction with other therapeutic agents. The fact that it is orally active makes it attractive not only as part of the initial treatment, but also as part of a maintenance regimen. Scale-up and formulation studies are currently underway. Future efforts will be focused on GLP toxicology and studies to determine dosing range, route, and schedule in support of an IND to advance these drugs into Phase I/II clinical trials. (Supported by grant 2R44CA14456 from the NCI and R01GM20818 from the NIH). Citation Format: Cathy A. Swindlehurst, Kyle W. H. Chan, Leah Fung, Robert W. Sullivan, Sabine Ottilie, Sergey V. Slepenkov, Shile Huang, Robert E. Rhoads. Oral multi-pathway inhibitors for the treatment of triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4527. doi:10.1158/1538-7445.AM2014-4527

Abstract B109: Novel orally bioavailable triple-negative breast cancer drug candidates that prevent the development of cancer associated fibroblasts in the tumor stroma

One in 8 women in the US will develop breast cancer during her lifetime. Breast cancer is the second most commonly diagnosed form of cancer and the second leading cause of cancer deaths in women. The main cause of breast cancer death is metastasis. Although the survival rate is 99% for localized disease, it is only 23% for distant stage disease. Changes in the tumor stroma create an environment that provides support for tumor growth, progression, invasion and metastasis. The most prominent cell type in the tumor stroma is an activated form of fibroblasts, known as cancer (or carcinoma) associated fibroblast (CAF). In breast cancer, as much as 80% of stromal fibroblasts acquire the CAF phenotype. Triple negative (estrogen receptor, progesterone receptor, and HER2 negative) breast cancer (TNBC) has a low detection rate by mammography, and TNBC tumors generally have a higher mean size and grade at diagnosis than other forms of breast cancer. TNBC can be particularly aggressive with a high rate of recurrence and metastasis and a less favorable prognosis than other subtypes (64% vs. 81%, respectively). Unlike other forms of breast cancer (Tamoxifen for ER+ tumors, Herceptin for tumors with HER2/Neu), no targeted therapy exists for TNBC. We have previously described small molecule inhibitors of translation initiation with safety and efficacy in animal models for the treatment of TNBC. One of these compounds, NM891, showed a durable response after treatment cessation even though plasma and tumor tissue concentrations were negligible. Recent data indicate that this may be a result of NM891 acting on the stroma that supports the growth and progression of the tumor. In preliminary studies, NM891 and NM922 (a related analog) were shown to prevent the TGF-β dependent activation of fibroblasts using alpha smooth muscle actin (α-SMA) as a biomarker of CAF. Studies to determine the effect of NM891 and NM922 on pathways (such as TGF-β) that drive the development of CAF are ongoing. In addition, both NM891 and NM922 have recently been demonstrated to be orally bioavailable (20.5% and 19.6%, respectively, after oral gavage compared to i.v. administration in mice) with reasonable serum half-lives (1.5 and 2.4 hours, respectively), making them attractive not only for initial treatment, but also as part of a maintenance regimen. Future studies include the development of formulations to further enhance oral bioavailability and serum half-life as well as determination of optimum dosing range and schedule in support of an IND. NM891 and NM922 are promising orally bioavailable clinical candidates that preferentially kill tumor cell and also prevent the transition of stimulated fibroblasts to the CAF phenotype, thus potentially decreasing recurrence and metastasis and increasing survival rates in TNBC. Citation Format: Leah Fung, Kyle W.H. Chan, Robert W. Sullivan, Sabine Ottilie, Cathy A. Swindlehurst. Novel orally bioavailable triple-negative breast cancer drug candidates that prevent the development of cancer associated fibroblasts in the tumor stroma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B109.