OT-82, a novel anticancer drug candidate that targets the strong dependence of hematological malignancies on NAD biosynthesis.

Effective treatment of some types of cancer can be achieved by modulating cell lineage-specific rather than tumor-specific targets. We conducted a systematic search for novel agents selectively toxic to cells of hematopoietic origin. Chemical library screenings followed by hit-to-lead optimization identified OT-82, a small molecule with strong efficacy against hematopoietic malignancies including acute myeloblastic and lymphoblastic adult and pediatric leukemias, erythroleukemia, multiple myeloma, and Burkitt's lymphoma in vitro and in mouse xenograft models. OT-82 was also more toxic towards patients-derived leukemic cells versus healthy bone marrow-derived hematopoietic precursors. OT-82 was shown to induce cell death by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD synthesis. In mice, optimization of OT-82 dosing and dietary niacin further expanded the compound's therapeutic index. In toxicological studies conducted in mice and nonhuman primates, OT-82 showed no cardiac, neurological or retinal toxicities observed with other NAMPT inhibitors and had no effect on mouse aging or longevity. Hematopoietic and lymphoid organs were identified as the primary targets for dose limiting toxicity of OT-82 in both species. These results reveal strong dependence of neoplastic cells of hematopoietic origin on NAMPT and introduce OT-82 as a promising candidate for the treatment of hematological malignancies.

p53 is a suppressor of inflammatory response in mice.

Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor kappaB (NFkappaB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF-inducible genes that are largely regulated by NFkappaB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFkappaB-dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild-type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53-deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFkappaB, plays the role of a general "buffer" of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFkappaB in tumors.

A small molecule inhibitor of p53 stimulates amplification of hematopoietic stem cells but does not promote tumor development in mice.

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could, in theory, contribute to the increased frequency of tumor development observed in p53-deficient mice and humans. In our previous work, we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice, PFTb impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition, we showed that PFTb stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly, however, PFTb treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus, although PFTb administration led to increased numbers of HSCs and HPCs, it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis.

Small molecule screening reveals a transcription-independent pro-survival function of androgen receptor in castration-resistant prostate cancer.

In prostate cancer (PCa) patients, initial responsiveness to androgen deprivation therapy is frequently followed by relapse due to development of treatment-resistant androgen-independent PCa. This is typically associated with acquisition of mutations in AR that allow activity as a transcription factor in the absence of ligand, indicating that androgen-independent PCa remains dependent on AR function. Our strategy to effectively target AR in androgen-independent PCa involved using a cell-based readout to isolate small molecules that inhibit AR transactivation function through mechanisms other than modulation of ligand binding. A number of the identified inhibitors were toxic to AR-expressing PCa cells regardless of their androgen dependence. Among these, some only suppressed PCa cell growth (ARTIS), while others induced cell death (ARTIK). ARTIK, but not ARTIS, compounds caused disappearance of AR protein from treated cells. siRNA against AR behaved like ARTIK compounds, while a dominant negative AR mutant that prevents AR-mediated transactivation but does not eliminate the protein showed only a growth suppressive effect. These observations reveal a transcription-independent function of AR that is essential for PCa cell viability and, therefore, is an ideal target for anti-PCa treatment. Indeed, several of the identified AR inhibitors demonstrated in vivo efficacy in mouse models of PCa and are candidates for pharmacologic optimization.

Small-molecule multidrug resistance-associated protein 1 inhibitor reversan increases the therapeutic index of chemotherapy in mouse models of neuroblastoma.

The multidrug resistance-associated protein 1 (MRP1) has been closely linked to poor treatment response in several cancers, most notably neuroblastoma. Homozygous deletion of the MRP1 gene in primary murine neuroblastoma tumors resulted in increased sensitivity to MRP1 substrate drugs (vincristine, etoposide, and doxorubicin) compared with tumors containing both copies of wild-type MRP1, indicating that MRP1 plays a significant role in the drug resistance in this tumor type and defining this multidrug transporter as a target for pharmacologic suppression. A cell-based readout system was created to functionally determine intracellular accumulation of MRP1 substrates using a p53-responsive reporter as an indicator of drug-induced DNA damage. Screening of small-molecule libraries in this readout system revealed pyrazolopyrimidines as a prominent structural class of potent MRP1 inhibitors. Reversan, the lead compound of this class, increased the efficacy of both vincristine and etoposide in murine models of neuroblastoma (syngeneic and human xenografts). As opposed to the majority of inhibitors of multidrug transporters, Reversan was not toxic by itself nor did it increase the toxicity of chemotherapeutic drug exposure in mice. Therefore, Reversan represents a new class of nontoxic MRP1 inhibitor, which may be clinically useful for the treatment of neuroblastoma and other MRP1-overexpressing drug-refractory tumors by increasing their sensitivity to conventional chemotherapy.

Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation.

p53-dependent apoptosis contributes to the side effects of cancer treatment, and genetic or pharmacological inhibition of p53 function can increase normal tissue resistance to genotoxic stress. It has recently been shown that p53 can induce apoptosis through a mechanism that does not depend on transactivation but instead involves translocation of p53 to mitochondria. To determine the impact of this p53 activity on normal tissue radiosensitivity, we isolated a small molecule named pifithrin-mu (PFTmu, 1) that inhibits p53 binding to mitochondria by reducing its affinity to antiapoptotic proteins Bcl-xL and Bcl-2 but has no effect on p53-dependent transactivation. PFTmu has a high specificity for p53 and does not protect cells from apoptosis induced by overexpression of proapoptotic protein Bax or by treatment with dexamethasone (2). PFTmu rescues primary mouse thymocytes from p53-mediated apoptosis caused by radiation and protects mice from doses of radiation that cause lethal hematopoietic syndrome. These results indicate that selective inhibition of the mitochondrial branch of the p53 pathway is sufficient for radioprotection in vivo.

p53 inhibitor pifithrin alpha can suppress heat shock and glucocorticoid signaling pathways.

Pifithrin alpha (PFTalpha) is a chemical compound isolated for its ability to suppress p53-mediated transactivation. It can protect cells from p53-mediated apoptosis induced by various stimuli and reduce sensitivity of mice to gamma radiation. Identification of molecular targets of PFTalpha is likely to provide new insights into mechanisms of regulation of p53 pathway and is important for predicting potential risks associated with administration of PFTalpha-like p53 inhibitors in vivo. We found that PFTalpha, in addition to p53, can suppress heat shock and glucocorticoid receptor signaling but has no effect on nuclear factor-kappaB signaling. PFTalpha reduces activation of heat shock transcription factor (HSF1) and increases cell sensitivity to heat. Moreover, it reduces activation of glucocorticoid receptor and rescues mouse thymocytes in vitro and in vivo from apoptotic death after dexamethasone treatment. PFTalpha affected both signaling pathways in a p53-independent manner. These observations suggest that PFTalpha targets some unknown factor that is common for three major signal transduction pathways.