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Video of caspase 3 activity used to capture images used in quantification of caspase activation, indicating apoptosis induction. Representative video of UC3 cells treated with APX2009 as red indicator dye generated from Caspase 3 activity is shown overlayed with brightfield image for each treatment. Duration of the video is the 72 hour duration of the experiment, as quantified and shown in Figure 2. Representative stills are shown in supplementary figure 3.

Confirmation of APE1/Ref-1 and STAT3 expression in 3d and monolayer conditions in RP-B-O1 and RP-B-O2 cells. Basal growth conditions as described in the methods for each cell line were used for monolayer and 3D conditions. No significant differences in expression relative to actin were observed in either condition. Representatuive blot is shown in [A], and quantified data as the ratio of each protein to actin from 3 determinations is shown in [B,C].

Video of caspase 3 activity used to capture images used in quantification of caspase activation, indicating apoptosis induction. Representative video of UC3 cells treated with DMSO control red dye by itself, Duration of the video is the 72 hour duration of the experiment, as quantified and shown in Figure 2. Representative stills are shown in supplementary figure 3.

Supplementary Figure S9 shows TIE2 and macrophage markers expression.

Video of caspase 3 activity used to capture images used in quantification of caspase activation, indicating apoptosis induction. Representative video of UC3 cells treated with APX2009 as red indicator dye generated from Caspase 3. Duration of the video is the 72 hour duration of the experiment, as quantified and shown in Figure 2. Representative stills are shown in supplementary figure 3.

Bladder cancer is the ninth most common cause of cancer-related deaths worldwide. Although cisplatin is used routinely in treating bladder cancer, refractory disease remains lethal for many patients. The recent addition of immunotherapy has improved patient outcomes; however, a large cohort of patients does not respond to these treatments. Therefore, identification of innovative molecular targets for bladder cancer is crucial. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in both DNA repair and activation of transcription factors through reduction–oxidation (redox) regulation. High APE1/Ref-1 expression is associated with shorter patient survival time in many cancer types. In this study, we found high APE1/Ref-1 expression in human bladder cancer tissue relative to benign urothelium. Inhibition of APE1/Ref-1 redox signaling using APE1/Ref-1–specific inhibitors attenuates bladder cancer cell proliferation in monolayer, in three-dimensional cultures, and in vivo. This inhibition corresponds with an increase in apoptosis and decreased transcriptional activity of NF-κB and STAT3, transcription factors known to be regulated by APE1/Ref-1, resulting in decreased expression of downstream effectors survivin and Cyclin D1 in vitro and in vivo. We also demonstrate that in vitro treatment of bladder cancer cells with APE1/Ref-1 redox inhibitors in combination with standard-of-care chemotherapy cisplatin is more effective than cisplatin alone at inhibiting cell proliferation. Collectively, our data demonstrate that APE1/Ref-1 is a viable drug target for the treatment of bladder cancer, provide a mechanism of APE1/Ref-1 action in bladder cancer cells, and support the use of novel redox-selective APE1/Ref-1 inhibitors in clinical studies.Significance:This work identifies a critical mechanism for APE1/Ref-1 in bladder cancer growth and provides compelling preclinical data using selective redox activity inhibitors of APE1/Ref-1 in vitro and in vivo.

Structures and NMR analysis of novel ref-1 inhibitors APX2009 and APX2014. A. APX3330; NMR and HPLC as previously published.15-18 B. APX2009; HPLC: HPLC were performed using an Alltech Alltima column C18 5u, 250 x 5.6 mm, flow 1 mL/min at 40 .C. Elution was with a mobile phase of 15:10:75 water:A1:methanol where A1 was made using 700 mL of water, 300 mL methanol and 3mL trimethylamine to which phosphoric acid was added to bring the pH to 3.4. HPLC analysis showed a purity of >99%. NMR: 300 MHz NMR -conforms to structure. NMR (CDCl3) Î' 8.15 (m, 2H), 7.75 (m,2H), 6.2 (s, 1H), 4.1 (s, 3H), 3.6 (br d, 4H), 2.2 (t, 2H), 1.4 (m, 4H), 1.25 (br d, 4H), 0.85(t, 3H). B. APX2014; HPLC: HPLC analysis was performed as for APX2009, but using a Zorbax SB Phenyl C18 5u, 250 x 4.6 mm column. Elution was with a mobile phase of 25:10:65 water:A1:methanol where A1 was made using 700 mL of water, 300 mL methanol and 3mL trimethylamine to which phosphoric acid was added to bring the pH to 3.4. Flow 1 mL/min at 40 .C. and showed a purity of >99%. NMR: 300 MHz NMR -conforms to structure. NMR (CDCl3) Î' 8.8 (br s,1H), 8.1 (m, 2H), 7.75 (m, 2H), 6.7 (s, 1H), 4.15 (s, 3H), 3.9 (s, 3H), 2.2 (m, 2H), 1.4 (m, 2H), 0.85 (t, 3H).

Confirmation of cell death induction by caspase activity assay and PARP cleavage. Representative images for caspase activity via red fluorescence [A] for the data calculated and shown in Figure 2, each with matching brightfield images. Representative examples for T24 and UC3 cells are shown form cells treated with the growth-inhibitor EC50 concentrations for each drug, in each cell line. PARP cleavage was used to confirm the induction of apoptotic mechanisms by western blotting for cleaved PARP1 [B], with representative cell experiments in which UC3 cells were treated with EC50 and EC90 concentrations of each drug.

Receptor tyrosine kinase inhibitors have shown clinical benefit in clear cell renal cell carcinoma (ccRCC), but novel therapeutic strategies are needed. The angiopoietin/Tie2 and MET pathways have been implicated in tumor angiogenesis, metastases, and macrophage infiltration. In our study, we used trebananib, an angiopoietin 1/2 inhibitor, and a novel small-molecule MET kinase inhibitor in patient-derived xenograft (PDX) models of ccRCC. Our goal was to assess the ability of these compounds to alter the status of tumor-infiltrating macrophages, inhibit tumor growth and metastases, and prolong survival. Seven-week-old SCID mice were implanted subcutaneously or orthotopically with human ccRCC models. One month postimplantation, mice were treated with angiopoietin 1/2 inhibitor trebananib (AMG 386), MET kinase inhibitor, or combination. In our metastatic ccRCC PDX model, RP-R-02LM, trebananib alone, and in combination with a MET kinase inhibitor, significantly reduced lung metastases and M2 macrophage infiltration (P = 0.0075 and P = 0.0205, respectively). Survival studies revealed that treatment of the orthotopically implanted RP-R-02LM tumors yielded a significant increase in survival in both trebananib and combination groups. In addition, resection of the subcutaneously implanted primary tumor allowed for a significant survival advantage to the combination group compared with vehicle and both single-agent groups. Our results show that the combination of trebananib with a MET kinase inhibitor significantly inhibits the spread of metastases, reduces infiltrating M2-type macrophages, and prolongs survival in our highly metastatic ccRCC PDX model, suggesting a potential use for this combination therapy in treating patients with ccRCC.

Legend for supplementary videos.

APE1/Ref-1 localization in human tumors. Additional examples of APE1/Ref-1 immunofluorescence in human bladder cancer specimens at 10X and 40X. Lower power (10X) [A] demonstrates transition from nuclear localized APE1/Ref-1 in the epithelial compartment (Epi) to cytosolic staining in the invasive regions of a human tumor, in the muscularis (Musc) in a single image, with high power (40x) views showing the cellular localization [B,C]. Additional examples in high power are shown in [D], from 3 different specimens (1-3), exclusively nuclear staining in the epithelial compartment (Epi) and cytosolic staining in the invasive satellite lesions (INV).

Animal body weights

Specificity of TFE3 antibody across TFE3 oncoproteins

A complete list of targeted genes in the PI3K/AKT signaling pathway with their associated miRNA from cluster 3.

Supplementary Figure Legend

List of chemokines/cytokines altered with PR/MR

Additional data on the role of PR/MR on tumor microenvironment

Purpose:Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease.Experimental Design:We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models.Results:The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3–tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation.Conclusions:These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3–tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3–tRCC.

Pie chart for TFE3 binding sites

Purpose:Diet and healthy weight are established means of reducing cancer incidence and mortality. However, the impact of diet modifications on the tumor microenvironment and antitumor immunity is not well defined. Immunosuppressive tumor-associated macrophages (TAMs) are associated with poor clinical outcomes and are potentially modifiable through dietary interventions. We tested the hypothesis that dietary protein restriction modifies macrophage function toward antitumor phenotypes.Experimental Design:Macrophage functional status under different tissue culture conditions and in vivo was assessed by Western blot, immunofluorescence, qRT-PCR, and cytokine array analyses. Tumor growth in the context of protein or amino acid (AA) restriction and immunotherapy, namely, a survivin peptide–based vaccine or a PD-1 inhibitor, was examined in animal models of prostate (RP-B6Myc) and renal (RENCA) cell carcinoma. All tests were two-sided.Results:Protein or AA-restricted macrophages exhibited enhanced tumoricidal, proinflammatory phenotypes, and in two syngeneic tumor models, protein or AA-restricted diets elicited reduced TAM infiltration, tumor growth, and increased response to immunotherapies. Further, we identified a distinct molecular mechanism by which AA-restriction reprograms macrophage function via a ROS/mTOR-centric cascade.Conclusions:Dietary protein restriction alters TAM activity and enhances the tumoricidal capacity of this critical innate immune cell type, providing the rationale for clinical testing of this supportive tool in patients receiving cancer immunotherapies.

Effect of different rapamycin concentrations

Enriched KEGG PI3K/AKT signaling pathway visualization

Gene list for Fig.5B, C, D

Pie chart for TFE3 binding sites

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

ChiP-seq for TFE3 in UOK-146

Schema for multimodal inhibition in tRCC

Supplementary Figure Legends

Supplemental data on additional models to support the role of EZH2 in RTKi resistance

Introduction The impact of lung parenchymal-only failure on patient survival after stereotactic ablative body radiotherapy (SABR) for early stage non-small cell lung cancer (NSCLC) remains unclear. Methods The study population included 481 patients with early stage NSC LC who were treated with 3 to 5 fraction SABR between 2000 and 2016. The primary study objective was to assess the impact of out-of-field lung parenchymal-only failure (OLPF) on overall survival (OS). Results At a median follow-up of 5.9 years, the median OS was 2.7 years for all patients. Patients with (OLPF) did not have a significantly different OS compared to patients who never failed (p=0.0952, median OS 4.1 years with failure vs. 2.6 years never failure). Analysis in a 1:1 propensity score-matched cohort for KPS, co-morbidity score, and smoking status showed no differences in OS between patients that never failed and those with OLPF (p=0.8). In subgroup analyses exploring impact of time of failure on OS, patients with OLPF 6 months or more after diagnosis did not have significantly different OS compared to those who never failed, when accounting for immortal time bias (p= 0.3, median OS 4.3 years vs 3.5 years never failure). Only seven patients in our dataset failed within 6 months of treatment, of which only four were confirmed to be true failures; therefore, limited data is available in our cohort on the impact of OLPF in ≤6 months on OS. Conclusions OLPF after SABR for early stage NSCLC does not appear to adversely affect OS, especially if occurring at least 6 months after SABR. More studies are needed to understand if OLPF within 6 months of SABR is associated with adverse OS. These data are useful when discussing prognosis of lung parenchymal failures after initial SABR.

Prostate cancer (PCa) is the most common cancer diagnosed in men in the Western countries. Epidemiological studies have suggested that environmental factors such as the Western diet, characterized by the consumption of high caloric food containing large amounts of animal protein and fats, play a key role in the pathogenesis of PCa. We propose that dietary intervention, by reducing caloric or protein intake, may be beneficial for the treatment of PCa. Our previous data suggests that dietary protein restriction reduces tumor growth in a LuCaP23.1 patient-derived xenograft (PDX) model of PCa. Thus, we tested the hypothesis whether caloric restriction induced by alternate-day fasting has a similar effect. Interestingly, caloric restriction had antitumor activity in the LuCaP23.1 model but only alternate-day fasting reduced the expression of androgen receptor (AR), a major driver of PCa growth. Therapies targeting AR activity, termed androgen deprivation therapies including the AR antagonist enzalutamide (ENZ), are the current standard of care for PCa. To address the therapeutic potential of combining alternate-day fasting with anti-androgens, we utilized the syngeneic mouse PCa xenograft models MyC-CaP and PTEN−/− RB−/−, and the human LuCaP23.1 PDX model. These AR-positive xenograft models harbor the oncogenic drivers most frequently found in PCa (i.e., MYC amplification and loss of the tumor suppressor PTEN). Here, we show that alternate-day fasting enhances the activity of ENZ in these PCa xenograft models. Interestingly, the combination of alternate-day fasting with ENZ treatment reduced AR expression and signaling, and decreased proliferation and tumor growth as compared to either single treatment alone. Consistent with these findings, the reduction of AR activity by the depletion of endogenous androgens following surgical castration was enhanced with alternate-day fasting. Conversely, dietary protein restriction had no effect in combination ENZ in vivo, suggesting that modulation of AR by alternate-day fasting enhances the effect of anti-androgens. Transcriptomic analysis of tumors from fasted mice confirmed decreased AR transcriptional activity and revealed that several nutrient sensitive pathways were reduced by this dietary regimen, including PI3K/AKT signaling, mTORC1 signaling, and glycolysis. Additionally, we observed that several known factors induced by fasting were upregulated in tumors from fasted mice, including SIRT1 which has been suggested to modulate AR transcriptional activity. In conclusion, we demonstrate that alternate-day fasting reduces AR signaling and enhances the activity of ENZ in several PCa xenograft models. Overall, this study suggests that caloric restriction may improve the efficacy of anti-androgen therapy in PCa patients. Citation Format: Ricardo Cordova, May Elbanna, Angela Klunk, Christopher Rupert, Li Shen, Yanqing Wang, David Goodrich, Ron Wek, Kirk Staschke, Luigi Fontana, Roberto Pili. Alternate-day fasting enhances the activity of anti-androgen therapy in prostate cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4239.

Receptor tyrosine kinase inhibitors have shown clinical benefit in clear cell renal cell carcinoma (ccRCC), but novel therapeutic strategies are needed. The angiopoietin/Tie2 and MET pathways have been implicated in tumor angiogenesis, metastases, and macrophage infiltration. In our study, we used trebananib, an angiopoietin 1/2 inhibitor, and a novel small-molecule MET kinase inhibitor in patient-derived xenograft (PDX) models of ccRCC. Our goal was to assess the ability of these compounds to alter the status of tumor-infiltrating macrophages, inhibit tumor growth and metastases, and prolong survival. Seven-week-old SCID mice were implanted subcutaneously or orthotopically with human ccRCC models. One month postimplantation, mice were treated with angiopoietin 1/2 inhibitor trebananib (AMG 386), MET kinase inhibitor, or combination. In our metastatic ccRCC PDX model, RP-R-02LM, trebananib alone, and in combination with a MET kinase inhibitor, significantly reduced lung metastases and M2 macrophage infiltration (P = 0.0075 and P = 0.0205, respectively). Survival studies revealed that treatment of the orthotopically implanted RP-R-02LM tumors yielded a significant increase in survival in both trebananib and combination groups. In addition, resection of the subcutaneously implanted primary tumor allowed for a significant survival advantage to the combination group compared with vehicle and both single-agent groups. Our results show that the combination of trebananib with a MET kinase inhibitor significantly inhibits the spread of metastases, reduces infiltrating M2-type macrophages, and prolongs survival in our highly metastatic ccRCC PDX model, suggesting a potential use for this combination therapy in treating patients with ccRCC.

Bladder cancer is the ninth most common cause of cancer-related deaths worldwide. Although cisplatin is used routinely in treating bladder cancer, refractory disease remains lethal for many patients. The recent addition of immunotherapy has improved patient outcomes; however, a large cohort of patients does not respond to these treatments. Therefore, identification of innovative molecular targets for bladder cancer is crucial. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in both DNA repair and activation of transcription factors through reduction–oxidation (redox) regulation. High APE1/Ref-1 expression is associated with shorter patient survival time in many cancer types. In this study, we found high APE1/Ref-1 expression in human bladder cancer tissue relative to benign urothelium. Inhibition of APE1/Ref-1 redox signaling using APE1/Ref-1–specific inhibitors attenuates bladder cancer cell proliferation in monolayer, in three-dimensional cultures, and in vivo. This inhibition corresponds with an increase in apoptosis and decreased transcriptional activity of NF-κB and STAT3, transcription factors known to be regulated by APE1/Ref-1, resulting in decreased expression of downstream effectors survivin and Cyclin D1 in vitro and in vivo. We also demonstrate that in vitro treatment of bladder cancer cells with APE1/Ref-1 redox inhibitors in combination with standard-of-care chemotherapy cisplatin is more effective than cisplatin alone at inhibiting cell proliferation. Collectively, our data demonstrate that APE1/Ref-1 is a viable drug target for the treatment of bladder cancer, provide a mechanism of APE1/Ref-1 action in bladder cancer cells, and support the use of novel redox-selective APE1/Ref-1 inhibitors in clinical studies. Significance: This work identifies a critical mechanism for APE1/Ref-1 in bladder cancer growth and provides compelling preclinical data using selective redox activity inhibitors of APE1/Ref-1 in vitro and in vivo.

Purpose: Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease. Experimental Design: We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models. Results: The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3–tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation. Conclusions: These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3–tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3–tRCC.

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

INTRODUCTION: For patients with brain metastases, palliative radiation therapy (RT) has long been a standard of care for improving quality of life and optimizing intracranial disease control. The duration of time between completion of palliative RT and patient death has rarely been evaluated. METHODS: A compilation of two prospective institutional databases encompassing April 2015 through December 2018 was used to identify patients who received palliative intracranial radiation therapy. A multivariate logistic regression model characterized patients adjusting for age, sex, admission status (inpatient versus outpatient), Karnofsky Performance Status (KPS), and radiation therapy indication. RESULTS: 136 consecutive patients received intracranial palliative radiation therapy. Patients with baseline KPS

Deregulation of the cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer yet inhibitors against these kinases currently show use in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and what may undermine such dependency is poorly understood. Here we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2-activation, involving as a critical mechanistic events loss of the CDK inhibitor p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Purpose: Diet and healthy weight are established means of reducing cancer incidence and mortality. However, the impact of diet modifications on the tumor microenvironment and antitumor immunity is not well defined. Immunosuppressive tumor-associated macrophages (TAMs) are associated with poor clinical outcomes and are potentially modifiable through dietary interventions. We tested the hypothesis that dietary protein restriction modifies macrophage function toward antitumor phenotypes. Experimental Design: Macrophage functional status under different tissue culture conditions and in vivo was assessed by Western blot, immunofluorescence, qRT-PCR, and cytokine array analyses. Tumor growth in the context of protein or amino acid (AA) restriction and immunotherapy, namely, a survivin peptide–based vaccine or a PD-1 inhibitor, was examined in animal models of prostate (RP-B6Myc) and renal (RENCA) cell carcinoma. All tests were two-sided. Results: Protein or AA-restricted macrophages exhibited enhanced tumoricidal, proinflammatory phenotypes, and in two syngeneic tumor models, protein or AA-restricted diets elicited reduced TAM infiltration, tumor growth, and increased response to immunotherapies. Further, we identified a distinct molecular mechanism by which AA-restriction reprograms macrophage function via a ROS/mTOR-centric cascade. Conclusions: Dietary protein restriction alters TAM activity and enhances the tumoricidal capacity of this critical innate immune cell type, providing the rationale for clinical testing of this supportive tool in patients receiving cancer immunotherapies.

Background: Data from epidemiological studies have linked dietary intake to the development of prostate cancer (CaP). We have previously shown that dietary protein restriction inhibits tumor growth by modulating PI3K/mTOR signaling in vivo. Additionally we have shown that dietary methionine restriction influence macrophage polarization which enhances the ability of the immune system to fight cancer. In this study we hypothesize that caloric restriction is capable of restricting tumor growth and potentiating the antitumor effect of androgen deprivation therapy (ADT) and PD1 inhibition. Methods: We utilized two prostate cancer models for our in vivo studies; castrate resistant LuCaP 23.1 AI prostate cancer model and MYC-Driven prostate cancer model in C57BL/6 mice. Caloric restriction was carried out by exposing mice to alternating fasting either as a single intervention (in LuCaP 23.1 model) or in combination with ADT (enzalutamide or surgical castration) or PD-1 inhibition in Myc-CaP model. Tumor sizes and weights were blindly assessed during the study and upon study termination respectively. IHC staining for both Ki-67 and mTOR phosphorylation was done to assess the impact of fasting+/- ADT on tumor growth and mTOR signaling. Macrophage polarization/distribution in tumors was assessed using immunofluorescence. Blood was collected at the end of the study for future comprehensive analysis of PBMCS. Results: In both models, alternating fasting was associated with significant decrease in tumor weight at the end of study in comparison to control group (*p Conclusions: Caloric restriction can hinder prostate cancer growth and potentiate the therapeutic effect of ADT. Our results provide basis for the translational use of dietary modification both as preventive measure as well as a therapeutic intervention that can improve the benefit of current standard treatments. Citation Format: May Elbanna, Justin Budka, Paige Dausinas, Remi Adelaiye-Ogala, Nur Damayanti, Ashley Orillion, Eri Banno, Luigi Fontana, Roberto Pili. Caloric restriction potentiates the therapeutic benefit of androgen deprivation therapy and alters macrophage polarization when combined with PD1 inhibition in murine models of 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 1611.

// Lei Wei 1,* , Sreenivasulu Chintala 2,5,7,* , Eric Ciamporcero 2,* , Swathi Ramakrishnan 2,5 , May Elbanna 2,7 , Jianmin Wang 1 , Qiang Hu 1 , Sean T. Glenn 3 , Mitsuko Murakami 4 , Lu Liu 4 , Eduardo Cortes Gomez 4 , Yuchen Sun 4 , Jacob Conroy 4 , Kiersten Marie Miles 4 , Kullappan Malathi 6 , Sudha Ramaiah 6 , Anand Anbarasu 6 , Anna Woloszynska-Read 2,5 , Candace S. Johnson 2,5 , Jeffrey Conroy 4 , Song Liu 1 , Carl D. Morrison 4 and Roberto Pili 2,7 1 Department of Biostatistics & Bioinformatics, Roswell Park Cancer Institute, Buffalo, NY, USA 2 Genitourinary Program, Roswell Park Cancer Institute, Buffalo, NY, USA 3 Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA 4 Center for Personalized Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 5 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA 6 Medical & Biological Computing Laboratory, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, INDIA 7 Genitourinary Program, Indiana University-Simon Cancer Center, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Roberto Pili, email: // Keywords : urothelial carcinoma, patient-derived xenograft, PI3KCA Received : October 15, 2016 Accepted : October 22, 2016 Published : November 03, 2016 Abstract Purpose: Effective systemic therapeutic options are limited for bladder cancer. In this preclinical study we tested whether bladder cancer gene alterations may be predictive of treatment response. Experimental design: We performed genomic profiling of two bladder cancer patient derived tumor xenografts (PDX). We optimized the exome sequence analysis method to overcome the mouse genome interference. Results: We identified a number of somatic mutations, mostly shared by the primary tumors and PDX. In particular, BLCAb001, which is less responsive to cisplatin than BLCAb002, carried non-sense mutations in several genes associated with cisplatin resistance, including MLH1 , BRCA2, and CASP8 . Furthermore, RNA-Seq analysis revealed the overexpression of cisplatin resistance associated genes such as SLC7A11 , TLE4, and IL1A in BLCAb001. Two different PIK3CA mutations, E542K and E545K, were identified in BLCAb001 and BLCAb002, respectively. Thus, we tested whether the genomic profiling was predictive of response to a dual PI3K/mTOR targeting agent, LY3023414. Despite harboring similar PIK3CA mutations, BLCAb001 and BLCAb002 exhibited differential response, both in vitro and in vivo . Sustained target modulation was observed in the sensitive model BLCAb002 but not in BLCAb001, as well as decreased autophagy. Interestingly, computational modelling of mutant structures and affinity binding to PI3K revealed that E542K mutation was associated with weaker drug binding than E545K. Conclusions: Our results suggest that the presence of activating PIK3CA mutations may not necessarily predict in vivo treatment response to PI3K targeted therapies, while specific gene alterations may be predictive for cisplatin response in bladder cancer models and, potentially, in patients as well.

Background: We have previously reported that two bladder cancer PDX models that carry distinct PIK3CA helical domain (HD) mutations respond differently to dual PI3K/mTOR inhibitor (LY3023414), where one model is sensitive (RP-B-02, PIK3CA E545K MUT) while the other is resistant (PIK3CA, E542K MUT) (Wei L et al Oncotarget 2016). Thus, we transduced these mutations in isogeneic cells to determine whether each mutation is indeed associated with a distinct oncogenic potential, as well as differential therapeutic response. Subsequently, we tested rational drug combinations capable of overriding resistance to PI3K targeted therapy (i.e. bromodomain inhibitor). Methods: Isogeneic HEK cells and urothelial cells (SV-HUC) were transduced with plasmids carrying either wild type (WT) or mutant PIK3CA (E545K and E542K mutants). Proliferation and growth were compared both in 2D and 3D culture respectively. We tested the therapeutic response to LY3023414 in 2D and 3D culture. Western blotting was used to test target modulation in the PI3K/mTOR pathway in response to treatment. JQ1 and LY3023414 combination studies were done in vitro. Calcusyn™ was used to determine whether synergy between the two agents exists in the context of either mutation. Results: PIK3CA E542K mutation has a growth advantage compared to the E545K mutation. This observation was more significant in 3D culture, which more faithfully represents the complexity of the tumor microenvironment. Isogeneic HEK cells expressing the E542K mutation were significantly more resistant to LY3023414 treatment both in 3D and 2D. This resonates with the resistance we initially reported in the bladder cancer PDX models carrying the E542K mutation. Interestingly, LY3023414+JQ1 combination was synergistic in the RP-B-01 (E542K MUT, but not in the RP-B-02 cell line (E545K MUT). Conclusion: PIK3CA mutation status is predictive of response to PI3K targeted therapy in bladder cancer, where PIK3CA E542K mutation confers resistance to PI3K targeted therapy. The synergistic combination of LY3023414 and the bromodomain inhibitor JQ1 can override resistance to PI3K targeted therapy. This drug combination may be of clinical significance in the context of PIK3CA E542K, but not E545K mutation. Citation Format: May Faek Elbanna, Nur Damayanti, Sreenivasulu Chintala, Roberto Pili. PIK3CA E542K mutation in bladder cancer confers resistance to PI3K targeted therapy but synergy with BET inhibition [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 1825.

Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite the identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathways resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we assessed the role of wild type TFE3 (wt-TFE3) in RCC cell bearing TFE3 fusion with genes associated with pre-mRNA splicing factor machinery (NONO, SFPQ and PRCC).Methods: Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft established in NSG mice. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. RNA interference (RNAi) mediated knockdown with differential exon targeting strategy was employed to study wt-TFE3 loss of function in RP-R07, UOK-109 and UOK-146, respectively. Real time monitoring of cell viability assay with multiplex readout were developed to investigate the effect of wt-TFE3 loss of function in 2D and 3D culture system. Two different 3D models were used;1) Tumor growth model, in which cancer cell interaction with extracellular matrix and stromal cells were represented in a multiculture-spheroid system; 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid, and the invasion trajectories were observed and quantified. Exogenous expression of wt-TFE3-EGFP was utilized to study the protein subcellular localization in RP-R07, UOK-109 and UOK-146 2D cultures and 3D culture system.Results: Consistent with the expression of chimeric TFE3, wt-TFE3-EGFP ectopic expression demonstrated strong nuclear localization in RP-R07, UOK-109 and UOK-146. Multiplex readout of cells viability assay showed that transient loss of wt-TFE3 in RP-R07, UOK-109 and UOK-146 curtails the proliferation rate of these cells in 2D and 3D models in fusion partner dependent manner (P Citation Format: Nur P. Damayanti, Khunsha Ahmed, May Elbanna, Chinghai Kao, Roberto Pili. Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes [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 3109.

Background: Cystine/glutamate exchanger xCT is a catalytic component of system xc- involved in transport of ‘conditionally indispensable’ amino acid cystine. Cystine transport is a rate limiting step for the synthesis of glutathione, a major intracellular redox regulator. Recently, we and other groups reported the overexpression of xCT and its association with drug resistance in several human cancers including bladder, glioma, breast, and colon. There are no studies to show the xCT expression in clear cell renal cell carcinoma (ccRCC) and its association with tyrosine kinase inhibitors resistance and metastasis. In the current study we have evaluated xCT expression in human ccRCC tumors arranged in tissue microarray (TMA) and determined its role in receptor tyrosine kinase inhibitor (RTKI) resistance and metastases using the patient derived tumor xenografts (PDX) models and TKI resistance ccRCC cells. Methods: Human Renal cell carcinoma tumor nephrectomy specimens arranged in tissue microarray (TMA) were used to determine xCT expression by immunohistochemistry. Patient derived tumor xenografts (PDX) of primary, metastasis, and sunitinib resistance were used to determine the role of xCT in RCC. To understand the molecular alterations associated with RTKI resistance, we have generated sunitinib resistance 786 OR ccRCC cells and performed RNAseq analysis. To determine the xCT inhibition effect on ccRCC tumor metastases, sulfasalazine, an inhibitor of xCT was used to treat metastatic ccRCC tumor xenografts transplanted in SCID mice. Results: Immunohistochemical evaluation of xCT in RCC TMA revealed that 70 % (19 out of 27) of the tumors express different levels of xCT. Association with tumor response to RTKI will be presented. RTKI less responsive PDX RP-R-02 was developed using the dose escalation treatment strategy and was found to have an upregulation of xCT when the tumors become less responsive to sunitinib. RNAseq analysis revealed differential gene expression of various pathway genes including lysosome biogenesis and function such as SLC7A11, HPS4, HPS5, CTSB, IFI30, PPT1, SCPEP1, TPP1, ATP6AP1L, MCOLN1, PRKAG2, and VPS18 in sunitinib resistant 786 OR cells compared to parental cells supports the role of lysosomes function in RTKI drug resistance. Furthermore, xCT inhibitor sulfasalazine treatment significantly decreased the metastasis lung nodules of RP-R-02LM in SCID mice demonstrated the xCT role in RCC tumor metastasis. Conclusions: We found preliminary evidence that overexpression of xCT may be associated with RTKI resistance in ccRCC. These results suggest that targeting the xCT in ccRCC may reverse the resistance and enhance the efficacy of RTKI. Additional studies using larger numbers of ccRCC tumors are required to identify xCT as a potential predictive biomarker for response/resistance to RTKI in ccRCC patients. Citation Format: Sreenivasulu Chintala, Remi Adelaiye-Ogala, Ashley Orillion, Sreevani Arisa, May Elbanna, Nur P. Damayanti, Roberto Pili. Association of xCT overexpression with RTKI resistance and metastases in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 94. doi:10.1158/1538-7445.AM2017-94

Background: Our previous work showed a significant reduction of tumor growth, macrophage infiltration, circulating IGF-1, and mTOR activation with low protein diet in a patient derived xenograft model of prostate cancer. The evolutionarily conserved, nutrient sensing, mTOR pathway plays a central role in both development of advanced stage prostate cancer and immune responsiveness. This study presents novel data on the impact of dietary protein modification on the function of the host immune system in response to prostate cancer and immunotherapy. Methods: Our In vitro studies utilized bone marrow or tumor derived (RP-B6 Myc) macrophages. In vivo studies utilized the recently characterized RP-B6 Myc model. Mice were fed ad libitum control or methionine restricted diets for four weeks prior to S.C. implantation with ~1mm2 tumor pieces. Treatment of survivin peptide vaccine (1mg/ml S.C. 1 X week) and anti-PD-1 (20mg/kg I.P. 2 X week) began at ~50mm2 tumor size. Tumor volumes were blindly recorded 2 X week. End point analyses include: tumor weight, flow cytometric analysis, proteomic profiler analyses, and microbiome analyses from each diet and treatment group. Results: We show here that while methionine restriction (MR) has little impact on our RP-B6 Myc prostate cancer cell line, it does yield a significant alteration in both the polarization and function of M1 and M2 macrophages. In the in vitro MR conditions, we observed significantly enhanced polarization of M1 macrophages and reduced polarization of M2 macrophages. Functional analysis revealed increased tumoricidal activity of both M1, ‘antitumor’, and M2, ‘pro-tumor,’ macrophages suggesting a flip in M2 function from tumor-promoting to tumoricidal. Further analysis of the released cytokines in MR media conditions yielded significant increase of antitumor cytokines & chemokines, such as IL-12, IL-27, CXCL9, CXCL10, CXCL11, CCL2, CCL4, and TNF-alpha, a double-edged sword which in our system correlates with decreased cancer cell viability upon co-culture with amino acid restricted M1 and M2 macrophages. Our preliminary results show dietary MR yielding a significant inhibition of prostate cancer growth in the RP-B6-Myc model and our ongoing study with survivin peptide vaccine and anti-PD-1 immunotherapies will be available to present in the conference. Conclusions: Our data suggest that restricting methionine is sufficient to alter both the polarization and tumoricidal function of macrophages. Our preliminarily results show that restricted dietary methionine is able to inhibit prostate cancer growth, modify the host immune response and better inhibit prostate cancer growth alone or in combination with immunotherapy. These results provide a strong basis to consider diet restriction as a means to limit cancer growth targeting tumor immune system and potentially to enhance cancer patient responses to immunotherapy. Citation Format: Ashley R. Orillion, Sreenivasulu Chintala, Remi Adelaiye-Ogala, Li Shen, Nur Damayanti, May Elbanna, Sreevani Arisa, Bennett Elzey, Chinghai Kao, Luigi Fontana, Roberto Pili. Methionine restriction increases macrophage tumoricidal activity and significantly inhibits prostate cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 250. doi:10.1158/1538-7445.AM2017-250

Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathway resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we sought to study the biology underlying oncogenesis in tRCC involving TFE3 fusion with genes associated with pre-mRNA splicing factor machinery; NONO, SFPQ and PRCC. Methods: To investigate the biology of different TFE3 fusion proteins, oncogenic phenotypes and oncogenic signaling were compared among cells with distinct endogenous expression of TFE3 fusions such as SFPQ-TFE3, NONO-TFE3, PRCC-TFE3. Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. TFE3 fusion transcripts were identified with RT-PCR. TFE3 fusion preferential DNA binding was assessed with ChIP-sequencing. Oncogenic phenotype assessment was done in 2D culture and two different 3D models; 1) Solid tumor model, in which cancer cell interaction with extracellular matrix and stromal cells, were represented in multiculture-spheroid system, 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid. Results: Dissimilar oncogenic phenotypes were seen among RP-R07, UOK-109 and UOK-146 in 2D and 3D culture. The presence of nuclear E-Cadherin was observed in all three cells model, with increasing level of nuclear expression in fusion partner dependent manner (P Conclusions: Our results indicate a distinct biology underlying tRCC oncogenesis in different TFE3 fusion models involving kinase reprograming and nucleocytoplasmic shuttling regulation. These differences also suggest the possibility of generating personalized treatments targeting specific TFE3 fusion genes associated with the spliceosome machinery. Citation Format: Nur P. Damayanti, Sreenivasulu Chintala, Ashley Orillion, Remi Adelaiye-Ogala, May F. Elbanna, Pete Hollenhorst, Roberto Pili. Delineating translocation renal cell carcinoma oncogenesis in cells harboring TFE3 fusion with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4475. doi:10.1158/1538-7445.AM2017-4475