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Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm (“secDrugs”) to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a ‘Double-Hit’ drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness–related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/‘scratch’ assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

Md Shakir Uddin Ahmed,1 Ahmad Bin Salam,1 Clayton Yates,1 Kyle Willian,2 Jesse Jaynes,3 Timothy Turner,4 Mohamed O Abdalla5 1Department of Biology, Tuskegee University, Tuskegee, 2Department of Chemistry and Biochemistry, Auburn University, Auburn, 3Department of Environmental Sciences, Tuskegee University, Tuskegee, AL, 4Department of Biology, Jackson State University, Jackson, MS, 5Department of Chemistry, Tuskegee University, Tuskegee, AL, USA Abstract: As an alternative therapeutic treatment to reduce or eliminate the current side effects associated with advanced prostate cancer (PCa) chemotherapy, a multifunctional double-receptor-targeting iron oxide nanoparticles (IONPs) (luteinizing hormone-releasing hormone receptor [LHRH-R] peptide- and urokinase-type plasminogen activator receptor [uPAR] peptide-targeted iron oxide nanoparticles, LHRH-AE105-IONPs) drug delivery system was developed. Two tumor-targeting peptides guided this double-receptor-targeting nanoscale drug delivery system. These peptides targeted the LHRH-R and the uPAR on PCa cells. Dynamic light scattering showed an increase in the hydrodynamic size of the LHRH-AE105-IONPs in comparison to the non-targeted iron oxide nanoparticles (NT-IONPs). Surface analysis showed that there was a decrease in the zeta potential values for drug-loaded LHRH-AE105-IONPs compared to the NT-IONPs. Prussian blue staining demonstrated that the LHRH-AE105-IONPs were internalized efficiently by the human PCa cell line, PC-3. In vitro, magnetic resonance imaging (MRI) results confirmed the preferential binding and accumulation of LHRH-AE105-IONPs in PC-3 cells compared to normal prostate epithelial cells (RC77N/E). The results also showed that LHRH-AE105-IONPs significantly maintained T2 MRI contrast effects and reduced T2 values upon internalization by PC-3 cells. These paclitaxel-loaded double-receptor-targeting IONPs also showed an approximately twofold reduction in PC-3 cell viability compared to NT-IONPs. Keywords: LHRH-R, uPAR, prostate cancer, iron oxide nanoparticles, targeted drug delivery

Background: Prostate cancer (PCa) is higher among African American (AA) than Caucasian Americans (CA). AAs are more frequently diagnosed with PCa with worse prognoses than CAs. Support of a genetic component to the high incidence and mortality rate in AA men is based on epidemiologic studies of men with similar genetic backgrounds. For instance, men in Ghana and Nigeria also have a high incidence of prostate cancer, as do men of African descent in the Caribbean islands and in the United Kingdom. Multiple reports indicate that PCa aggressiveness is the result of cancer stem cells (CSC). Furthermore, people of African Ancestry have been reported to have increased stemness gene signature in multiple tumor types. CD133, which is a well-known progenitor/stem cells marker, and is associated with prostate tumor relapse and metastasize due to the existence of prostate cancer stem cells (PCaCSC) has been well characterized, however, its role in prostate cancer racial disparity is unknown. Therefore, our objective is to analyze the significance of CD133 as a disparity stem cell biomarker in AA and CA patients with late-stage PCa. Methods: TGCA Prostate Dataset was used for patient data. The R and R studio statistical software was used for the analysis, and for the survival analysis, Log-Rank test was used to determine survival. H & E was used to determine the presence of prostate cancer and tumors were scored using Gleason grading. CD133 protein location and expression was determined by IHC, and quantified using Aperio imaging software. Results: We analyzed genomic data in TGCA for correlation between CD133 over-expression and worst patient survival outcome. Survival analysis indicated a poorer survival outcome in the AA with high CD133 expression during late-stage PCa compare to the CA with the same stage of PCa. Furthermore, the addition of CD44 furthermore increased the significant difference between the stem cell population (p= 0.04) between AA and CA patients. To confirm these results, we evaluated CD133 expression in (Gleason =7) PCa tumor from AA and CA patients and compared them to BPH. As in the TCGA data, we observed increased CD133 expression in AA patients. Conclusion: Elevated CD133 shows an association with patients with African Ancestry. Further studies are necessary on a larger series of cases to elucidate the role of CD133 in the development and progression of prostate cancer and its suitability as a prognostic biomarker. Citation Format: Benjamin Adu-Addai, Ahmad B. Salam, Ruksana Amin, Honghe Wang, Huixian Lin, Anghesom Ghebremedhin, Jason White, Jesse M. Jaynes, Clayton Yates. CD133 as a disparity stem cell biomarker in African Americans with late-stage prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-181.

Background: Tumor associated M-ϕs (TAMs) have a complex etiology as a result of soluble factors secreted by prostate tumor cells, creating an immunosuppressive micro-environment (M-E) for the progression of late stage PC that has metastasized to the bone. The result is an M2-like polarization that expresses a number of M2-TAM specific markers, including CD206. Therapeutic targeting of TAMs has proven to be a challenge, due partly to a lack of understanding of the function of this biomarker and the bi-directional effects of TAMs on tumor cells. Our objective is to characterize the CD206 receptor in PC patients, and to test the potential of a novel engineered CD206 targeted peptide (T-P) to reverse the immune-suppressive M-Env in late stage PC. Methods: The TGCA Prostate Dataset was used for patient data and R software was used for informatics analysis. Small-angle X-ray scattering (SAXS) was used to generate the NMR structure of CD206. Peptide binding to the naïve CD206 receptor was determined in silico using PIPER, and was validated by Surface Plasmon Resonance (SPR). In vivo efficacy of CD206 T-P was determined in mice inoculated with PC-3 intratibially. Mice were treated with peptide alone, daily or twice a week at 10, 20, and 50mg/Kg, as a monotherapy or in combination with 20mg/Kg Docetaxel (DTX) over a 21day period. CD86 (M1) and CD206/CD163 (M2-TAM) markers and pro-inflammatory cytokines were determined by FACS. Bone osteolysis was determined by IHC of the entire bone, and bone turnover was determined by IHC analysis of RANKL. Results: TCGA data showed that over-expression of CD206 in Human PC is significantly associated with poor survival. In silico analyses showed the T-P exhibited strong affinity towards CD206 and was validated by SPR (Kd =3.57 µM). In bone-metastatic xenograft models of CRPC (PC-3), the flux differential of relative fluorescence in IVIS imaging showed peptide alone (at 20mg/kg qD sQ) decreased fluorescence by 64.36% compared with the untreated group. FACS analyses of M1/M2 markers CD86/CD206 in mice treated with peptide showed a 33.87 fold decrease in CD206 (M2-TAM) expression and a 7.6 fold increase in CD86 (M1-TAM) expression, compared with only a 4.4 fold decrease in CD206 expression and 0.44 fold decrease in CD86 expression in DTX treated mice. Stem cell marker CD133 showed a 4.7 fold decrease expression compared to the untreated group further suggesting that TAM repolarization reversed the immunosuppressive M-E. X-ray of tumors in bone suggests a reduction of osteolysis and was confirmed by IHC staining and analysis of whole bone showing fibrous and inflammatory remodeling patterns of tumor regression in the peptide only group. Conclusion: These results suggest that the novel engineered CD206 T-P repolarizes M2-TAM phenotype to M1-TAM which reverses the immunosuppressive cancer M-E in late stage PC. Citation Format: Ahmad B. Salam, Benjamin Adu-Addai, Huixian Lin, Anghesom Ghebremedhin, Jason White, Ruksana Amin, Balasubramanyam Karanam, George Martin, Charles Garvin, Henry Lopez, Jesse M. Jaynes, Clayton Yates. Novel synthetic peptide targets CD206 receptor promoting the repolarization of prostate cancer (PCa) TAMs and increase the efficacy of conventional prostate cancer therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4062.

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Due to the harsh side effects of conventional chemo- and radiotherapies, targeted drug delivery is now gaining the focus of cancer researchers. The key for success of any newly developed targeted drug delivery systems is the novel design of targeting peptides which have high binding affinities to differentially over-expressed receptors on the surface of PCa cells. Two over-expressed such receptors, which can be targeted to the cell surface of PCa cells are: Gonadotropin Releasing Hormone Receptor (GnRH-R), and Prostate Specific Membrane Antigen (PSMA). The analysis done from molecular dynamics simulation data gave an important information about each interaction; such as RMSD, The number of contacts between receptor and peptide within a cutoff distance of 5 Å and the binding free energies, ΔGGBSA of the interactions. In all systems, the back bone RMSD deviation of the receptor was calculated using the last 10 ns of the sampling process and averaged over all trajectories. The RMSD data deviated between 1.22 - 2.51 Å. The free binding energies or ΔGGBSA was calculated from the last 10 ns of the sampling process. The QH1 system has the more negative ΔGGBSA -36.15 kcal mol-1 and the QH4 system has the least negative ΔGGBSA of -18.4 kcal mol-1 among the eight systems of GnRH-R. QH2 system exhibits a highly interactive hydrogen bond (bond distance is 2.65 Å) between 134th position of the receptor amino acid sequence GLU and 5th position of the qh2 peptide's TYR. The ΔGGBSA is for the qh2 system is – 34.92 kcal mol-1. In the PSMA and its targeting peptide interactions, T2IA has the least negative ΔGGBSA of -47.37 kcal mol-1. A salt bridge was observed between the 10th position of the T2IA peptide sequence ARG and the 328 position of the 3RBU receptor's GLU. Based on our molecular modeling studies, we have determined the best targeting peptides among all the newly designed peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated surface plasmon resonance (SPR) in near future. Citation Format: Ahmad Salam, Vincent Hembrick, Jesse Jaynes, Timothy Turner, Mohamed Abdalla. Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2170.