Paul Kwon

Selective growth inhibition of tumor cells by a novel histone deacetylase inhibitor, NVP-LAQ824.

We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48–72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G2-M phase of the cell cycle after compound treatment. However, an increased sub-G1 population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.

Activity of deacetylase inhibitor panobinostat (LBH589) in cutaneous T-cell lymphoma models: Defining molecular mechanisms of resistance.

Panobinostat (LBH589) is a highly potent deacetylase inhibitor that has demonstrated clinical efficacy in patients with advanced cutaneous T‐cell lymphoma (CTCL). To gain a better understanding of the compound activity in this tumor type, we investigated the cellular and molecular effects of panobinostat using both in vitro and in vivo models of CTCL. All 4 tested CTCL cell lines exhibited very high sensitivity to panobinostat‐induced growth inhibition. However, only 2 of 4 lines exhibited significant response to the cytotoxic activity of panobinostat. In a CTCL xenograft mouse tumor model, panobinostat treatment resulted in complete tumor regression. The difference in cell sensitivity to panobinostat‐induced death enabled us to further investigate potential mechanisms responsible for tumor sensitivity or resistance. In CTCL cell lines that were insensitive to panobinostat‐induced apoptosis, constitutively activated NF‐κB and high levels of Bcl‐2 were observed. Inhibition of Bcl‐2 sensitized cells to the cytotoxic activity of panobinostat. Conversely, knockdown of Bax diminished the CTCL cell sensitivity. Interestingly, panobinostat could induce cytotoxicity in vorinostat‐resistant CTCL cells by downregulating phosphorylated STAT3 and STAT5 proteins. These studies suggest distinct mechanisms responsible for resistance to different deacetylase inhibitors. We show that the intrinsic apoptotic signaling plays an essential role in mediating panobinostat anticancer activity. Moreover, cancer cell sensitivity to panobinostat treatment may be further improved by combination with inhibition of anti‐apoptotic factors. These data provide preclinical support that panobinostat, as a single agent or in combination with other anticancer agents, is a promising therapy for CTCL.

Optimization of the in Vitro Cardiac Safety of Hydroxamate-Based Histone Deacetylase Inhibitors

Histone deacetylase (HDAC) inhibitors have shown promise in treating various forms of cancer. However, many HDAC inhibitors from diverse structural classes have been associated with QT prolongation in humans. Inhibition of the human ether a-go-go related gene (hERG) channel has been associated with QT prolongation and fatal arrhythmias. To determine if the observed cardiac effects of HDAC inhibitors in humans is due to hERG blockade, a highly potent HDAC inhibitor devoid of hERG activity was required. Starting with dacinostat (LAQ824), a highly potent HDAC inhibitor, we explored the SAR to determine the pharmacophores required for HDAC and hERG inhibition. We disclose here the results of these efforts where a high degree of pharmacophore homology between these two targets was discovered. This similarity prevented traditional strategies for mitigating hERG binding/modulation from being successful and novel approaches for reducing hERG inhibition were required. Using a hERG homology model, two compounds, 11r and 25i, were discovered to be highly efficacious with weak affinity for the hERG and other ion channels.

Preclinical Antitumor Activity of a Novel Anti–c-KIT Antibody–Drug Conjugate against Mutant and Wild-type c-KIT–Positive Solid Tumors

Purpose: c-KIT overexpression is well recognized in cancers such as gastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC), melanoma, non–small cell lung cancer (NSCLC), and acute myelogenous leukemia (AML). Treatment with the small-molecule inhibitors imatinib, sunitinib, and regorafenib resulted in resistance (c-KIT mutant tumors) or limited activity (c-KIT wild-type tumors). We selected an anti–c-KIT ADC approach to evaluate the anticancer activity in multiple disease models. Experimental Design: A humanized anti–c-KIT antibody LMJ729 was conjugated to the microtubule destabilizing maytansinoid, DM1, via a noncleavable linker (SMCC). The activity of the resulting ADC, LOP628, was evaluated in vitro against GIST, SCLC, and AML models and in vivo against GIST and SCLC models. Results: LOP628 exhibited potent antiproliferative activity on c-KIT–positive cell lines, whereas LMJ729 displayed little to no effect. At exposures predicted to be clinically achievable, LOP628 demonstrated single administration regressions or stasis in GIST and SCLC xenograft models in mice. LOP628 also displayed superior efficacy in an imatinib-resistant GIST model. Further, LOP628 was well tolerated in monkeys with an adequate therapeutic index several fold above efficacious exposures. Safety findings were consistent with the pharmacodynamic effect of neutropenia due to c-KIT–directed targeting. Additional toxicities were considered off-target and were consistent with DM1, such as effects in the liver and hematopoietic/lymphatic system. Conclusions: The preclinical findings suggest that the c-KIT–directed ADC may be a promising therapeutic for the treatment of mutant and wild-type c-KIT–positive cancers and supported the clinical evaluation of LOP628 in GIST, AML, and SCLC patients. Clin Cancer Res; 24(17); 4297–308. ©2018 AACR.

Organismal aging and phosphorylation of transcription factors

Publisher Summary This chapter discusses the organismal aging and phosphorylation of transcription factors. Aging is a universal phenomenon for all living organisms and helps define the individual understanding of mortality and finality. Experimental data suggest that both genetic and epigenetic factors contribute to organismal aging. In addition, there is strong evidence linking molecular mechanisms in cellular senescence with aging and deciphering some of these mechanisms revealed differential gene expression levels between “old” and “young” cells. This variation in gene expression is found to result from altered transcription factor functions arising from differential phosphorylation states. Several important transcription factors thought to regulate cell cycle control and mediate responses to internal and external stimuli are discussed in the chapter. These factors include SRF, p62TCF, tumor suppressors, and NF- κ B. Considering that these transcription factors integrate and effect different signaling pathways, the consequence of aberrant phosphorylation states leading to the misregulation and/or disruption of the “normal” signaling cascade is significant. Therefore, unveiling the cellular activities of these factors is critical in understanding the aging process and other diseases, where stringent transcriptional regulation needs are to be maintained.