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Background.Targeting HER2 gene amplification is one of the great achievements in oncology resulting in the use of a wide array of anti-HER2 agents in the clinic. Unfortunately, 20% of patients still relapse with secondary organ metastasis and are currently incurable. While only 1.6% of primary non-HER2-amplified ER+ breast cancers harbor HER2 mutations, 6-10% of all metastatic breast cancers harbor HER2 mutations, suggesting their causal role in contributing to metastasis. The clinical value of HER2 activating mutations is being tested with the pan-HER tyrosine kinase inhibitor neratinib in phase II clinical trials (NCT01670877, NCT01953926, and NCT02465060). To date, neratinib has elicited only modest responses in ER+ breast cancer, often rapidly followed by progression. This study characterizes HER2-mutant induced resistance to endocrine therapy or neratinib induced resistance and resulting metastasis, in order to determine a more effective rational therapeutic approach for treating ER+ HER2-mutant breast cancer. Methods.HER2 mutation frequency and its impact on patient outcome was determined using METABRIC primary breast cancer (BC) and MSK IMPACT metastatic BC ER+ sequencing studies. Effects of estrogen (E2), fulvestrant, or neratinib on cell growth and HER2 signaling were examined on ER+/ILC cells (MM134) stably expressing HER2/WT, HER2/S310F, and HER2/L755S. Cell growth was measured using CellTiter-Glo and HER2 signaling was analyzed by western blot analysis. Additionally, the effect of these ER+/ILC and IDC HER2 mutations on tumor growth and endocrine or neratinib treatment resistance was determined using fat pad injections and MIND xenografts in NOD-scid gamma mice. Results.We searched ER+ sequencing datasets and identified HER2 mutations that are highly enriched in ER+ ILC as compared to ER+ IDC. These activating HER2 mutations in ER+ ILC are associated with early relapse and poor overall survival. Moreover, we are finding that ILC patients harboring the recurrent HER2/L755S mutation have worse overall survival compared to non-mutant HER2 ILC. MM134 cells expressing HER2/S310F and HER2/L755S show increased cell growth, strongly activated autophosphorylation of HER2, and increased downstream signaling (pMAPK and pAKT) as compared to cells expressing HER2/WT upon treatment with fulvestrant (1μM). Three clinically relevant in vivo models including ILC HER2/L755S Mammary INtraDuctal (MIND) xenografts, IDC HER2/L755S fat pad xenografts, and IDC HCI-003 (an ER+ patient-derived xenograft (PDX) naturally harboring the exon 20 activating HER2G778_P780 dup) exhibit fulvestrant and neratinib resistance and lung and ovary metastases. In addition, we find that HER2 mutations induced mTOR signaling. In contrast, however, the pan-HER drug poziotinib does potently inhibit tumor growth and organ-specific metastasis and perturbs mTOR activation in these models. Conclusion.We demonstrate that clinically associated HER2 mutations drive endocrine therapy or neratinib resistance and poor patient outcome in ER+ patients. Our data propose the use of the irreversible pan-HER TKI poziotinib for treating endocrine therapy or neratinib refractory ER+ HER2-mutant metastatic breast cancer. Citation Format: Shyam. M Kavuri, Rashi Kalra, Ching Hui Chen, Junkai Wang, Ahmad Bin Salam, Lacey Dobrolecki, Alaina Lewis, Christina Sallas, Clayton Yates, Carolina Gutierrez, Balasubramanyam Karanam, Meenakshi Anurag, Bora Lim, Matthew Ellis. Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ HER2 mutant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-24.

Supplementary Data from Poziotinib Inhibits HER2-Mutant–Driven Therapeutic Resistance and Multiorgan Metastasis in Breast Cancer

The pan-HER tyrosine kinase inhibitor (TKI) neratinib is therapeutically active against metastatic breast cancers harboring activating HER2 mutations, but responses are variable and often not durable. Here we demonstrate that recurrent HER2 mutations have differential effects on endocrine therapy responsiveness, metastasis, and pan-HER TKI therapeutic sensitivity. The prevalence and prognostic significance may also depend on whether the HER2 mutant has arisen in the context of lobular versus ductal histology. The most highly recurrent HER2 mutant, L755S, was particularly resistant to neratinib but sensitive to the pan-HER TKI poziotinib, alone or in combination with fulvestrant. Poziotinib reduced tumor growth, diminished multiorgan metastasis, and inhibited mTOR activation more effectively than neratinib. Similar therapeutic effects of poziotinib were observed in both an engineered HER2L755S MCF7 model and a patient-derived xenograft harboring a HER2G778_P780dup mutation. Overall, these findings support the need for clinical evaluation of poziotinib for the treatment of HER2-mutant metastatic breast cancer.Significance:Evaluation of the functional impact of HER2 mutations on therapy-induced resistance and metastasis identifies robust antitumor activity of poziotinib and supports the clinical evaluation of poziotinib in ER+ HER2 mutant breast cancer.

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.

Metastatic prostate cancer is the second leading cause of cancer deaths in US men. Resistance to standard medical castration and secondary taxane-based chemotherapy is nearly universal. Further, presence of cancer stem-like cells (EMT/epithelial to mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/advanced/lethal variants of PCa (AVPC).In this study, first we used single-cell RNA sequencing (scRNAseq) analysis to demonstrate that ARlow PCa cells in metastatic prostate cancer, including castration-sensitive tumors, harbored signatures of EMT, and ‘cancer stemness’. Next, we introduced a novel pharmacogenomics data-driven computational approach and identified several potential agents that can be re-purposed as novel secondary drugs (“secDrugs”) to treat advance variants of Prostate cancer. Using scRNAseq as a biomarker-based drug screen, we demonstrated that a majority of the single-cell subclones in mCRPC and mCSPC cell lines also showed significantly high expression of the NAMPT pathway genes, indicating that the secDrug FK866, which targets NAMPT, is potentially effective against drug-resistant and stem-cell-like subpopulation cluster. Next, we showed significant in vitro cytotoxicity of FK866 as single-agent and in combination with the taxanes or Enzalutamide against models of clinically-advanced PCa. We performed bulk- and single-cell RNAseq to identify several pathways underlining FK866 mechanism of action and found 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 performed a microfluidic chip-based cell migration assay that demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using multiple PCa patient datasets, we showed that FK866 is potentially capable of reversing expression of several genes associated with biochemical recurrence and inter-ethnic differences, including IFITM3 and LTB4R.Thus, using FK866 as a proof-of-concept drug, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

African American (AA) women with breast cancer are more likely to have higher inflammation and a stronger overall immune response, which correlate with poorer outcomes. In this report, we applied the nanostring immune panel to identify differences in inflammatory and immune gene expression by race. We observed a higher expression of multiple cytokines in AA patients compared to EA patients, with high expression of CD47, TGFB1, and NFKB1 associated with the transcriptional repressor Kaiso. To investigate the mechanism associated with this expression pattern, we observed that Kaiso depletion results in decreased expression of CD47, and its ligand SIRPA. Furthermore, Kaiso appears to directly bind to the methylated sequences of the THBS1 promotor and repress gene expression. Similarly, Kaiso depletion attenuated tumor formation in athymic nude mice, and these Kaiso-depleted xenograft tissues showed significantly higher phagocytosis and increased infiltration of M1 macrophages. In vitro validation using MCF7 and THP1 macrophages treated with Kaiso-depleted exosomes showed a reduced expression of immune-related markers (CD47 and SIRPA) and macrophage polarization towards the M1 phenotype compared to MCF7 cells treated with exosomes isolated from high-Kaiso cells. Lastly, analysis of TCGA breast cancer patient data demonstrates that this gene signature is most prominent in the basal-like subtype, which is more frequently observed in AA breast cancer patients.

The pan-HER tyrosine kinase inhibitor (TKI) neratinib is therapeutically active against metastatic breast cancers harboring activating HER2 mutations, but responses are variable and often not durable. Here we demonstrate that recurrent HER2 mutations have differential effects on endocrine therapy responsiveness, metastasis, and pan-HER TKI therapeutic sensitivity. The prevalence and prognostic significance may also depend on whether the HER2 mutant has arisen in the context of lobular versus ductal histology. The most highly recurrent HER2 mutant, L755S, was particularly resistant to neratinib but sensitive to the pan-HER TKI poziotinib, alone or in combination with fulvestrant. Poziotinib reduced tumor growth, diminished multiorgan metastasis, and inhibited mTOR activation more effectively than neratinib. Similar therapeutic effects of poziotinib were observed in both an engineered HER2L755S MCF7 model and a patient-derived xenograft harboring a HER2G778_P780dup mutation. Overall, these findings support the need for clinical evaluation of poziotinib for the treatment of HER2-mutant metastatic breast cancer. Significance: Evaluation of the functional impact of HER2 mutations on therapy-induced resistance and metastasis identifies robust antitumor activity of poziotinib and supports the clinical evaluation of poziotinib in ER+ HER2 mutant breast cancer.

Bortezomib and the other licensed 20S proteasome inhibitors show robust activity against liquid tumors like multiple myeloma, but have disappointed against solid tumors including ovarian cancer. Consequently, interest is mounting in alternative non-peptide based drugs targeting the proteasome’s 19S regulatory particle subunit, including its ubiquitin receptor RPN13. RA183 and RA375 are more potent analogs of the prototypic inhibitor of RPN13 (iRPN13) called RA190, and they show promise for the treatment of ovarian cancer. Here we demonstrate that rendering these candidate RPN13 inhibitors chiral and asymmetric through the addition of a single methyl to the core piperidone moiety increases their potency against cancer cell lines, with the S-isomer being more active than the R-isomer. The enhanced cancer cell cytotoxicities of these compounds are associated with improved binding to RPN13 in cell lysates, ATP depletion by inhibition of glycolysis and mitochondrial electron chain transport, mitochondrial depolarization and perinuclear clustering, oxidative stress and glutathione depletion, and rapid accumulation of high molecular weight polyubiquitinated proteins with a consequent unresolved ubiquitin proteasome system (UPS) stress response. Cytotoxicity was associated with an early biomarker of apoptosis, increased surface annexin V binding. As for cisplatin, BRCA2 and ATM deficiency conferred increased sensitivity to these iRPN13s. Ubiquitination plays an important role in coordinating DNA damage repair and the iRPN13s may compromise this process by depletion of monomeric ubiquitin following its sequestration in high molecular weight polyubiquitinated protein aggregates. Indeed, a synergistic cytotoxic response was evident upon treatment of several ovarian cancer cell lines with either cisplatin or doxorubicin and our new candidate iRPN13s, suggesting that such a combination approach warrants further exploration for the treatment of ovarian cancer.

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-β1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-β1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206(high) patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.

The loss of miR-200 family, through DNA methylation, results in cancer cells undergoing an epithelial to mesenchymal transition (EMT), and metastasis. In this study, we established that the transcriptional repressor Kaiso directly binds methylated regions of the miR-200 family, and this is reversed with 5-aza treatment. sh-Kaiso PC-3 cells display increased miR-200-a/b/c, miR-141, and miR-429 expression, with miR-200c demonstrating the most significant increase. Interestingly, overexpression of EGFR or treatment with EGF decreases miR-200c expression and this is reversed after treatment with EGFR specific kinase inhibitor PD153035. However, EGF did not have a significant effect on miR-200c in sh-Kaiso DU-145 or PC-3 cell lines, suggesting Kaiso silences miR-200c through the activation of EGFR signaling. Overexpression of Kaiso in LNCaP cells results in decreased expression of miR-200-a/b/c, miR-141, and miR-429, along with increased expression of ZEB1, p-EGFR and total EGFR levels. Overexpression of miR200c in PC-3 cells results in decreased expression of EGFR, ZEB1, ERK1/2 and Kaiso. Additionally, sh-Kaiso PC-3 demonstrates reduced in vivo tumor formation and metastasis. Thus, our data suggests that EGFR signaling regulates the silencing of miR-200 family through Kaiso binding to methylated regions in the promoter.

Background: Over 60% of the United States population is infected with herpes simplex virus 1 (HSV-1). Current HSV-1 treatment regimens exert their antiviral effects through a common mechanism of action and suffer from high dosing frequencies, which may contribute to patient noncompliance and the subsequent development of antiviral resistance. Although primarily known for their functions in maintaining homeostatic control of arterial blood and osmotic pressures, components of the Renin-Angiotensin Aldosterone System (RAAS) have been implicated in viral replication and some components demonstrated antiviral properties. However, the antiviral properties of RAAS components have not been well characterized in reference to HSV-1. Methods: To address this gap in knowledge, we evaluated the antiviral effects of captopril, an Angiotensin Converting Enzyme 1 (ACE-1) inhibitor, on HSV-1 infection in SH-SY5Y neuroblastoma cells. We demonstrated that captopril attenuates HSV-1-induced cytopathic effects (CPE) via cell-based and morphological assays. To investigate the potential mechanism, we conducted molecular modeling studies and identified the ability of captopril to interact with and bind HSV-1 glycoprotein D. To determine where in the virus life cycle captopril exerts its protective effects, we performed experiments observing the effect of captopril on both viral entry and replication utilizing a green fluorescent protein-tagged virus and subsequent quantitative Polymerase Chain Reaction. Results: Results suggest captopril protects cells from HSV-1-induced CPE through its effect on viral replication by increasing cell viability in infected cells and decreasing virus replication, which we propose is modulated through the decreased expression of ICP0. Conclusions: Collectively, the results presented here support further evaluation of captopril, and other RAAS components, as a basis for potential novel therapeutic interventions for the treatment of HSV-1, its associated pathologies, and potentially other virus infections.

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.

Purpose: 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 radio-therapies, targeted drug delivery is now gaining focus of cancer researchers. The key for success for 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. Four over-expressed receptors which can be targeted on the cell surface of PCa cells are: (GnRH), (EGFR), (PSMA), and (uPAR). Thus, we propose to study the binding affinity of tweleve novel designed targeting peptides (three peptides per receptor) for these over-expressed receptors. Initially, we will study the binding affinity using a computerized molecular modeling program. We will corroborate our modeling data by determining the dissociation constants of the peptide-receptor interaction using sensitive experimental tools such as miscroscale thermophoresis. Methods: The newly designed peptides were generated in pdb (protein database) format and the known pdb files of the receptors were collected from protein data bank. Cluspro 2.0, developed by Boston University, is a protein docking server with a molecular modeling program that was used to calculate the binding energies of these peptides to their corresponding receptors. The data obtained was analyzed using Pymol. Results: The molecular docking studies showed that the GnRH tarteting peptide QH10, uPAR targeting peptide CV13, EGFR targeting peptide RL10, and PSMA targeting peptide TH12 have the lowest mean binding energies for their respective top three binding conformations. The calculatd binding energies for the aftermentioed peptides-receptors combination are: -1285 KCal/M, -1029 KCal/M, -1019 KCal/M and -1037 KCal/M, respectively. There results suggested a very high interaction for these newly designed peptides and their corroponding receptors as the typical calculated binding energy for an antigen-antibody interaction is -700 KCal/M. Conclusion: Based on our molecular docking studies, we have determined the best targeting peptides among all the newly desiged peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated using microscale thermophoresis. This work is supported by grant funding from NIH/RCMI G12MD00758 (T. Turner) and NIH/NCI U54 CA118623 (T. Turner & M. Abdalla). Note: This abstract was not presented at the meeting. Citation Format: Mohamed O. Abdalla, Ahmad Bin Salam, Vincent Hembrick, Manikanthan Bhavaraju, Clayton Yates, Jesse Jaynes, Timothy Turner. Molecular modeling studies of novel receptor targeted peptides in the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2015-3694