CXCR1 and CXCR2 chemokine receptors and their ligands (CXCL1/2/3/7/8) play an important role in tumor progression. Tested to date CXCR1/2 antagonists and chemokine-targeted antibodies were reported to affect malignant cells in vitro and in animal models. Yet, redundancy of chemotactic signals and toxicity hinder further clinical development of these approaches. In this pre-clinical study we investigated the capacity of a novel small molecule dual CXCR1/2 inhibitor, Ladarixin (LDX), to attenuate progression of experimental human melanomas. Our data showed that LDX-mediated inhibition of CXCR1/2 abrogated motility and induced apoptosis in cultured cutaneous and uveal melanoma cells and xenografts independently of the molecular defects associated with the malignant phenotype. These effects were mediated by the inhibition of AKT and NF-kB signaling pathways. Moreover, systemic treatment of melanoma-bearing mice with LDX also polarized intratumoral macrophages to M1 phenotype, abrogated intratumoral de novo angiogenesis and inhibited melanoma self-renewal. Collectively, these studies outlined the pre-requisites of the successful CXCR1/2 inhibition on malignant cells and demonstrated multifactorial effects of Ladarixin on cutaneous and uveal melanomas, suggesting therapeutic utility of LDX in treatment of various melanoma types.

Bone marrow suppression due to exposure to ionizing radiation is a significant clinical problem associated with radiation therapy as well as with nonmedical radiation exposure. Currently, there are no small molecule agents available that can enhance hematopoietic regeneration after radiation exposure. Here, we report on the effective mitigation of acute hematopoietic radiation syndrome in mice by the synthetic triterpenoid, RTA 408. The administration of a brief course of RTA 408 treatment, beginning 24 h after lethal doses of radiation to bone marrow, significantly increased overall survival. Importantly, treatment with RTA 408 led to the full recovery of steady state hematopoiesis with normalization of the frequency of hematopoietic stem and progenitor cells. Moreover, hematopoietic stem cells from RTA 408-mitigated mice showed lineage-balanced, long-term, multilineage potential in serial transplantation assays, indicative of their normal self-renewal activity. The potency of RTA 408 in mitigating radiation-induced bone marrow suppression makes it an attractive candidate for potential clinical use in treating both therapy-related and unanticipated radiation exposure.

Normal tissue toxicity markedly reduces the therapeutic index of genotoxic anti-cancer agents including ionizing radiation. Countermeasures against tissue damage caused by radiation are limited by their potential to also protect malignant cells and tissues. Here we tested a panel of signal transduction modifiers for selective radioprotection of normal but not tumor tissues. These included three inhibitors of GSK3 (LiCl, SB216763 and SB415286) and two inhibitors of NF-κB (ethyl pyruvate and RTA 408). Among these, the thiol reactive triterpenoid RTA 408 emerged as a robust and effective protector of multiple organ systems (gastrointestinal, skin and hemopoietic) against lethal doses of radiation. RTA 408 preserved survival and proliferation of crypt cells in lethally irradiated small intestines while reducing apoptosis incidence in crypts and villi. In contrast, RTA 408 uniformly inhibited growth of established CWR-22Rv1, LNCaP/C4-2B, PC3 and DU145 xenografts either alone or combined with radiation. Anti-tumor effects in vivo were associated with reduced proliferation and intratumoral apoptosis and with inhibition of NF-κB-dependent transcription in PC3 cells. Selective protection of normal tissue compartments by RTA 408 critically depended on tissue context and could not be replicated in vitro. Collectively, these data highlight the potential of RTA 408 as a cytoprotective agent that may be safely used in chemoradiation approaches.

<p>Supplementary Figure 4: Effects of RTA 408 on radiation sensitivity of prostate cancer cell lines as determined by clonogenic survival assays. Cells were treated at 24 and 1 h with RTA 408 prior to IR exposure as indicated. Differences between curves were analyzed by two-way ANOVA, designating drug treatment and IR as variables. P value summaries of pair-wise comparisons between drug and vehicle treatment curves are shown.</p>

<p>Supplementary Figure 3: Effects of RTA 408 on prostate cancer cell survival in vitro. Cells were treated for 24 h with RTA 408 at different concentrations. Protein extracts were prepared from attached and/or detached cells to capture the full extent of apoptosis. Protein extracts from detached cells were prepared at RTA 408 concentrations {greater than or equal to}0.5 μM; solid lines above protein concentrations indicate detached cells as a source of proteins. Levels of cleaved caspase 3, cleaved PARP-1, cleaved cytokeratin-18 (M30) and cleaved caspase-8 were determined by immunoblot analyses.</p>