Human Patient-derived Xenograft Models Research Articles

A Compound That Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Nonspecific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small-molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell-cycle, metabolic, and enzymatic assays were used to demonstrate their mechanism of action. A human patient-derived xenograft model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small-molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

Abstract 614: Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices <1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 614.

Abstract 742: PYX-201, a stroma-targeting ADC composed of an anti-EDB+FN antibody conjugated to Auristatin0101, demonstrates strong anti-tumor efficacy across multiple human cancer indications in pre-clinical PDX tumor models

Abstract Stroma is crucial to support solid tumor growth, metastasis and resistance to treatment. While most antibody-drug conjugates (ADCs) directly bind to cancer cells, PYX-201, an investigational drug, is designed to target tumor stroma by binding to the extra-domain B splice variant of fibronectin (EDB+FN), a matrix protein abundantly expressed in the tumor microenvironment of many solid tumors with absent or low expression in normal adult tissues. In vitro studies demonstrated PYX-201 was strongly cytotoxic to EDB+FN positive Caki2 cells, but not to EDB+FN negative HT29 cells. In addition, PYX-201 was 30-times more potent than a non-targeted ADC control, demonstrating PYX-201 cytotoxic effects were EDB+FN dependent. Furthermore, when Caki2 and HT29-luciferase cancer cells were co-cultured, the luciferase activity was used to quantify the amount of EDB+FN negative HT29 cells only. Interestingly, PYX-201 was able to kill EDB+FN negative cancer cells in a co-culture assay as shown by a decrease in luciferase activity, demonstrating PYX-201 bystander activity. Pre-clinical mouse model studies were conducted to evaluate PYX-201 anti-tumor efficacy and pharmacokinetic (PK) properties. First, a comprehensive patient-derived xenograft (PDX) mouse model trial was designed to evaluate the anti-tumor efficacy of PYX-201. To fully represent the diversity and characteristics of human tumors, PDX models were pre-selected from ten solid tumor indications based on different levels of EDB+FN protein expression and stromal density. PYX-201 (Q4D × 4 at 3mg/kg) was well tolerated, and strong anti-tumor responses (% Tumor Growth Inhibition [TGI] >90%) were observed in PDX models across various cancer indications. Potential correlations between EDB+FN protein expression with anti-tumor activity will be evaluated. Remarkably, long-term anti-tumor efficacy of PYX-201 was observed as tumors did not relapse even after >100 days in a breast cancer PDX model. Additionally, plasma samples were analyzed from a cell line derived xenograft mouse model after a single dose of PYX-201 to determine its PK profile. At 3 mg/kg, the maximum concentration (Cmax) was 28.1 μg/mL, area under the curve (AUC) was 1,732.7 h*μg/mL, with a low clearance of 1.5 mL/h/kg, and the half-life of the mAb from PYX-201 was calculated at 81.2 h. These PK parameters were associated with strong PYX-201 anti-tumor efficacy (% TGI >90%) in the H1975 xenograft model. Altogether, PYX-201 was well tolerated and demonstrated strong anti-tumor efficacy in a variety of pre-clinical human PDX models. PYX-201 is a promising and innovative ADC which is currently under investigation in a Phase I clinical trial (NCT05720117). Citation Format: Nicolas Severe, Amanda Facklam, Liz McMichael, Biplab Das, Sara Lewandowski, Justin Trickett, Liyang Diao, Chengfeng Merriman, Chuan Shen, Jianwen Feng, Shawn Harriman, Marsha Crochiere, Philipp Steiner, Jan Pinkas. PYX-201, a stroma-targeting ADC composed of an anti-EDB+FN antibody conjugated to Auristatin0101, demonstrates strong anti-tumor efficacy across multiple human cancer indications in pre-clinical PDX tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 742.

A first-in-class pan-lysyl oxidase inhibitor impairs stromal remodeling and enhances gemcitabine response and survival in pancreatic cancer.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.

Abstract P6-11-10: Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer

Abstract BACKGROUND: Nearly 50% of patients with triple negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NACT) retain residual tumors resulting in high rates of metastatic relapse and poor overall survival. Residual tumors surviving NACT (Adriamycin plus cyclophosphamide; AC) were found to undergo a metabolic transition to heightened mitochondrial oxidative phosphorylation (oxphos; PMID: 30996079). Pharmacologic inhibition of mitochondrial electron transport chain (ETC) complex I with IACS-010759 (PMID: 29892070) had enhanced efficacy in residual, rather than treatment-naïve, tumors of orthotopic patient-derived xenograft (PDX) models. Our analyses of mitochondrial structure and function in human TNBC cell lines revealed differing adaptations in residual cells surviving treatment with conventional NACT agents. While DNA-damaging chemotherapies (e.g.Adriamycin, carboplatin) induced mitochondrial fusion and oxphos, taxanes (e.g.paclitaxel, docetaxel) induced mitochondrial fragmentation and reduced oxphos (Baek et al., Biorxiv Doi 10.1101/2022.02.25.481996). The mechanistic basis of these mitochondrial adaptations is not yet understood. The mitochondrial ETC consists of 92 proteins, 13 of which are encoded in the mitochondrial genome (mtDNA) and translated by the mitoribosome, while the remaining are encoded by the nuclear genome (nDNA), translated by the cytoribosome, and inserted into the inner mitochondrial membrane by accessory proteins, namely Oxidase (Cytochrome C) Assembly 1-Like (OXA1L). Disruption of OXA1L in mammalian cells has been shown to affect the levels and activity of ETC complexes I, III, IV, and V, and thus diminish oxphos. We aim to determine whether mitochondrial translation and OXA1L activity represent therapeutic vulnerabilities to overcome pro-survival metabolic adaptations in chemoresistant TNBC thereby augmenting treatment response. METHODS: Weare evaluating the effects of conventional TNBC chemotherapies singly, and in standard combinations, on mitochondrial translation and ETC formation in human TNBC cells and PDX models(PIM001-P, WHIM14, BCM15116) using metabolomic and proteomic profiling. To perturb these processes genetically, we knocked down (KD) OXA1Lwith siRNA. We are complementing these studies pharmacologically using conventional antibiotics, such as tigecycline, as previous studies showed they inhibit mitochondrial translation in breast and other cancers (PMID: 25625193). These studies will reveal whether OXA1L and mitochondrial translation are required for metabolic adaption and chemotherapy resistance of residual TNBC cells. PDX preclinical trials based on our published residual tumor testing schema (PMID: 30996079), will reveal whether the sequential combination of NACT followed by tigecycline can effectively perturb residual tumor relapse. RESULTS: Proteomic profiling of longitudinally harvested PDX tumors demonstrates substantial disruption of mitochondria-and nuclear-encoded ETC components in residual vs. treatment-naïve tumors. Interestingly, these patterns are distinct between different chemotherapy treatments, with an increase of ETC components in carboplatin-treated residual tumors compared to a decrease in docetaxel-treated residual tumors. Western blot analyses of human cell lines show OXA1LKD perturbs levels of both nuclear-and mitochondria-encoded ETC components. Preliminary findings suggest OXA1LKD increases sensitivity to chemotherapies in human TNBC cell lines. Finally, tigecycline effectively inhibits TNBC cell growth. We next will evaluate whether residual cells not killed by conventional chemotherapies have enhanced tigecycline susceptibility. CONCLUSION: These data suggest targeting mitochondrial translation may be a promising approach to overcome pro-survival metabolic adaptations in residual TNBC cells not killed by conventional chemotherapies. Citation Format: Mariah J. Berner, Lily Baek, Junegoo Lee, Philip L. Lorenzi, Mei Leng, Alexander B. Saltzman, Anna Malovannaya, Lacey E. Dobrolecki, Christina Sallas, Michael T. Lewis, Gloria V. Echeverria. Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-10.

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance. MethodsTaking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry. ResultsThe success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90–100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4+ T cells were significantly reduced. ConclusionsThrough the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.

Dll1-Mediated Notch Signaling Drives Tumor Cell Cross-talk with Cancer-Associated Fibroblasts to Promote Radioresistance in Breast Cancer.

Dll1+ breast cancer cells activate Notch signaling in cancer-associated fibroblasts that increases Wnt ligand secretion and leads to β-catenin-driven radioresistance and metastasis, opening new therapeutic avenues for breast cancer.

Predictive modeling of resistance to SMO inhibition in a patient-derived orthotopic xenograft model of SHH medulloblastoma.

BackgroundInhibition of the sonic hedgehog (SHH) pathway with Smoothened (SMO) inhibitors is a promising treatment strategy in SHH-activated medulloblastoma, especially in adult patients. However, the problem is that tumors frequently acquire resistance to the treatment. To understand the underlying resistance mechanisms and to find ways to overcome the resistance, preclinical models that became resistant to SMO inhibition are needed.MethodsTo induce SMO inhibitor resistant tumors, we have treated a patient-derived xenograft (PDX) model of SHH medulloblastoma, sensitive to SMO inhibition, with 20 mg/kg Sonidegib using an intermitted treatment schedule. Vehicle-treated and resistant models were subjected to whole-genome and RNA sequencing for molecular characterization and target engagement. In vitro drug screens (76 drugs) were performed using Sonidegib-sensitive and -resistant lines to find other drugs to target the resistant lines. One of the top hits was then validated in vivo.ResultsNine independent Sonidegib-resistant PDX lines were generated. Molecular characterization of the resistant models showed that eight models developed missense mutations in SMO and one gained an inactivating point mutation in MEGF8, which acts downstream of SMO as a repressor in the SHH pathway. The in vitro drug screen with Sonidegib-sensitive and -resistant lines identified good efficacy for Selinexor in the resistant line. Indeed, in vivo treatment with Selinexor revealed that it is more effective in resistant than in sensitive models.ConclusionsWe report the first human SMO inhibitor resistant medulloblastoma PDX models, which can be used for further preclinical experiments to develop the best strategies to overcome the resistance to SMO inhibitors in patients.

Alisertib Synergistically Strengthens the Anti-Leukemia Activity of Venetoclax in TCF3-Hlf B-ALL

Background: Despite maximally-intensive chemotherapy and stem cell transplantation, survival of patients with the very rare t(17;19)/TCF3-HLF B-acute lymphoblastic leukemia (B-ALL) subtype remains effectively 0%. Prior studies have demonstrated association of the oncogenic TCF3-HLF fusion protein with multi-drug resistance via increased expression of ABC and P-glycoprotein drug efflux transporters, as well as via upregulation of pro-survival Ras and BCL-2 pathways. Preclinical studies and small clinical case series of targeted inhibitor addition to chemotherapy or antibody-based and cellular immunotherapies have aimed to improve outcomes for children with TCF3-HLF ALL. Unfortunately, targeting of these activated pathways with the BCL-2 inhibitor venetoclax or other small molecule inhibitors (SMIs) has been minimally or only transiently effective, suggesting more complex mechanism(s) of chemoresistance. In recent years, many patients with relapsed TCF3-HLF ALL have enrolled on clinical trials of CD19- or CD22-targeted immunotherapies. However, TCF3-HLF ALL frequently harbours deactivating mutations in PAX5, a major B-cell regulator and indispensable CD19 transcription factor, placing immunotherapy-treated patients at higher risk of CD19 antigen-loss relapse. New therapies remain needed to prevent relapse and attempt cure.Methods: We designed an unbiased kinome-wide CRISPR/Cas9 library to identify essential drivers in TCF3-HLF leukemogenesis. We screened the human TCF3-HLF ALL cell line HAL-01 and our TCF3-HLF ALL patient-derived xenograft (PDX) model ALL1807 (Hurtz JCI 2020, Schultz Genome Biol 2021), then validated identified targets using 49 SMIs targeting receptor tyrosine kinases (RTK), MEK signaling, and cell cycle pathways. We selected promising candidate inhibitor pairings with non-overlapping mechanisms of action and assessed for in vitro drug synergy via SynergyFinder analyses. Finally, we assessed the in vivo activity of targeted inhibitors in ALL1807 and two newly established TCF3-HLF ALL PDX models (CPCT-0002, CPCT-0003) created from primary pediatric specimens obtained via the LEAP Consortium (Pikman Cancer Disc 2021).Results: RNA-sequencing of HAL-01 and ALL1807 cells followed by functional protein association (STRING) analysis confirmed a network of significantly upregulated (>3-fold) plasma membrane and cytoplasm components of RTK pathways as well as BCL-2. The intersection of the results of the SMI drug library screen with the top 1% targets identified in CRIPSR/Cas9 screen determined p120-RasGAP and Aurora kinase A (AURKA) as therapeutic targets in TCF3-HLF ALL. In vitro treatment of HAL-01 or ALL1807 cells with the RasGAP inhibitor, pluripotin, or the AURKA inhibitor, alisertib, across a range of concentrations demonstrated robust anti-ALL activity. AURKA and RasGAP co-immunoprecipitated and this protein complex was disrupted with alisertib or pluripotin treatment. The AURKB inhibitor barisertib had minimal activity against TCF3-HLF ALL cells, confirming preferential dependency of these cells upon AURKA. Treatment of TCF3-HLF ALL cells with the BCL-2i venetoclax did not disrupt the AURKA/RasGAP complex, suggesting its different mechanism of action and potential for combinatorial drug therapy. Next, we found that alisertib and venetoclax synergistically killed TCF3-HLF ALL cells. Finally, we observed superior inhibition of in vivo leukemia with dual AURKA and BCL-2 inhibitor treatment of three TCF3-HLF ALL PDX models compared to single-agent alisertib or venetoclax (Figure 1).Conclusions: We identified AURKA as a critical new driver in TCF3-HLF ALL via orthogonal genetic and functional assays and confirmed prior observations of BCL-2 dependency in our models. We validated these key targets via in vitro and in vivo pharmacologic inhibition studies with drug synergy detected with combined alisertib and venetoclax in human TCF3-HLF ALL cell lines and PDX models. We posit that dual AURKA and BCL-2 inhibition is a clinically-pragmatic and potentially effective therapeutic strategy for patients with this rare, but highly fatal, leukemia subtype that merits formal clinical investigation. [Display omitted] DisclosuresCarroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding. Tasian: Incyte Corporation: Research Funding; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy.

Comparison of Induction Chemotherapy in NSG and NOD- Rag1 null IL2rg null Mouse Models of FLT3 Mutant AML

Background and objectives: Induction chemotherapy that combines Ara-C (cytarabine, 7 days) with an anthracycline such as daunorubicin or doxorubicin (Adriamycin, 3 days) (DA) is a standard induction therapy in AML. Immunodeficient NSG or NSGS mice are extremely useful for preclinical drug testing and are promiscuous recipients of primary AML engraftment. However, these models have limitations for use of genotoxic chemotherapy that often induces toxicity and mortality to the recipient mice, particularly in AML patient-derived xenograft (PDX) models when irradiation is required. The sensitivity of NSG or NSGS mice to genotoxic drugs and irradiation are due to SCID mutation in the mice. In this study we have compared tolerability and efficacy of DA chemotherapy in NSG and NRG (NOD- Rag1 null IL2rg null) AML-bearing mice. We used a FLT3 mutant AML Molm13 cell line to generate AML xenograft in NRG and NSG mice and then tested the response to chemotherapy using combined Ara-C and doxorubicin chemotherapy with or without sub-lethal irradiation. Testing of chemotherapy regimen in human AML PDX models is ongoing and will be presented.Methods and results: The AML mouse models were established by i.v. injection of GFP and firefly luciferase transduced Molm13 cells (0.6 x 106 cells per mouse) into NSG and NRG mice (n=5 per group, F, 8 weeks old). In experiments testing sub-lethal radiation, NSG mice were irradiated with 200 cGy and NRG mice with 400 cGy before cell injection. AML cell engraftment and growth in vivo was monitored by bioluminescent imaging (BLI). Engraftment was confirmed on day-3 (Fig. 1A-B) and chemotherapy started on day-4 using combined Ara-C (50 mg/kg, i.v. 5 days) and doxorubicin (1.5 mg/kg, i.v., 3 days) (DA 5+3) or PBS. NSG and NRG mice (n=3 each) that served as naïve controls without bearing Molm13 cells also received the chemotherapy. Mouse health conditions and body weights were monitored daily, and mice were followed for survival.As shown by BLI in Figure 1 A-B, DA 5+3 significantly reduced leukemic burden in both NRG and NSG mice compared to PBS treated mice (p<0.001 on day 13 or 14). The chemotherapy also induced transient toxicity in all treated mice with significant body weight loss in the sub-lethal irradiated AML NSG mice (Figure 1C, 30% loss on day 10, p=0.016 vs PBS treated mice), resulting in lethality earlier than PBS treated mice (Figure 1D, p=0.0019). Complete blood cell counts showed pancytopenia with anemia of the moribund treated Molm13 NSG mice. Overall, chemotherapy induced more body weight loss in NSG than that in NRG mice (a transient 25% body weight loss in naive NSG and AML NSG mice compared to 10% loss in NRG mice on day 10), indicating that NSG mice are more susceptible to chemotherapy-induced toxicity. All AML NRG mice and NSG mice without prior sub-lethal irradiation survived the chemotherapy and had significantly prolonged survival.We further challenged the NRG mice with a more intensive chemotherapy regimen by administrating Ara-C for 7 days (DA 7+3) mimicking the one used in AML patients. BLI confirmed more effective leukemia control by this regimen compared to DA 5+3 and significantly extended survival.Conclusion: The results demonstrate that Molm13 bearing NSG and NRG mice have identical latency, leukemia growth kinetics, and survival, suggesting that like NSG mice, NRG mice are suitable recipients for AML xenograft studies. Both Molm13 NSG mice and NRG mice showed strong efficacy in response to chemotherapy using Ara-C (cytarabine, 50 mg/kg) and doxorubicin (1.5 mg/kg) with the regimen of DA 5+3. However, this regimen was not tolerable when mice required prior sublethal irradiation, with excessive bodyweight loss and mortality in NSG but not in NRG mice. In contrast, Molm13 NRG mice can tolerate a more intensive combination chemotherapy regimen of AraC administered for 7 days (DA 7+3). In summary, NRG mouse model showed better tolerance to chemotherapy and irradiation than the NSG model, thus representing a superior in vivo AML model for pre-clinical studies with genotoxic agents. [Display omitted] DisclosuresCarter:Daiichi Sankyo: Research Funding; novartis: Research Funding.

Abstract 4558: Antitumor activity of AMG757, a half-life extended (HLE) bispecific T-cell engager (BiTE®) immune therapy targeting DLL3, in human PDX and orthotopic mouse models of small cell lung cancer (SCLC)

Abstract SCLC is a highly aggressive neuroendocrine tumor with poor prognosis and limited therapeutic options. Delta-like ligand 3 (DLL3) is a tumor-associated antigen that is highly specific for SCLC; expression is elevated in tumors but minimal and mainly cytoplasmic in normal tissues. AMG 757 is an HLE BiTE® immune therapy that is designed to redirect T-cell cytotoxicity to cancer cells by binding to DLL3 on the surface of cancer cells and CD3 on T cells. AMG 757 is being evaluated in a phase 1 clinical trial in patients with relapsed/refractory SCLC (NCT03319940). We evaluated the efficacy and pharmacodynamic (PD) effects of AMG 757 in three preclinical models of SCLC that express DLL3. We used the LXFS 1129 patient-derived xenograft (PDX) model to assess AMG 757 efficacy in established, subcutaneously implanted tumors similar to human SCLC tumors with their associated stroma. We also developed two orthotopic models of SCLC (SHP-77 and H82) to assess AMG 757 efficacy and PD at biologically relevant sites. In the SHP-77 orthotopic model, cells injected intravenously trafficked to the lungs where they formed diffuse tumors similar to primary SCLC tumors. In the H82 orthotopic model, cells injected intravenously formed discrete metastatic lesions in the liver, mimicking a major site for SCLC metastasis. In each model, mice bearing established tumors received a single infusion of human T cells and were then dosed with AMG 757 or a control HLE BiTE molecule. In the LXFS 1129 PDX model, treatment with AMG 757 induced complete tumor regression in 8 out of 10 mice, whereas treatment with the control HLE BiTE molecule showed no tumor growth inhibition. In the orthotopic SHP-77 model, treatment with AMG 757 led to significant tumor growth inhibition in the lungs relative to treatment with a control HLE BiTE molecule; the bioluminescence (BLI) signal decreased to near the limit of detection 22 days after start of treatment. AMG 757 antitumor activity was associated with increased T-cell trafficking and expansion in tumors; a single dose of AMG 757 significantly increased the number of human CD4+ and CD8+ cells in lungs as assessed by flow cytometry. In the orthotopic H82 model, AMG 757 treatment completely cleared visible lesions from the liver, as assessed by a decrease in BLI to near-background levels and macroscopic analysis of the liver tissue. AMG 757 antitumor activity was associated with increased CD8+ T-cell infiltration as assessed by flow cytometry and IHC and upregulation of the T cell surface activation markers CD25, CD69, PD-1, and 4-1BB as assessed by flow cytometry. Together, these preclinical data demonstrate that AMG 757 can recruit and engage T cells to tumors, consistent with the BiTE® mechanism of action, and that AMG 757-mediated redirected T-cell lysis can drive significant antitumor activity in established SCLC tumor models. Citation Format: Keegan Cooke, Juan Estrada, Jinghui Zhan, Jonathan Werner, Sean Caenepeel, Mike Giffin, Julie M. Bailis, Angela Coxon, Paul E. Hughes, Jude Canon. Antitumor activity of AMG757, a half-life extended (HLE) bispecific T-cell engager (BiTE®) immune therapy targeting DLL3, in human PDX and orthotopic mouse models of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4558.

Abstract 1060: Genetic and pharmacologic inhibition of Skp2, an E3 ubiquitin ligase and RB1-target, has antitumor activity in RB1-deficient human and mouse small cell lung cancer (SCLC)

Abstract The identification of driver mutations and their corresponding targeted drugs has led to significant improvements in the treatment of non-small cell lung cancer (NSCLC) and other solid tumors; however, similar advances have not been made in the treatment of small cell lung cancer (SCLC). Due to their aggressive growth, frequent metastases, and resistance to chemotherapy, the five-year overall survival of SCLC is less than 5%. While SCLC tumors can be sensitive to first-line therapy of cisplatin and etoposide, most patients relapse, often in less than 3 months after initial therapy. Dozens of drugs have been tested clinically in SCLC, including more than 40 agents that have failed in phase III trials. The near uniform bi-allelic inactivation of the tumor suppressor gene RB1 is a defining feature of SCLC. RB1 is mutated in highly aggressive tumors, including SCLC, where its functional loss, along with that of TP53, is both required and sufficient for tumorigenesis. While it is known that RB1 mutant cells fail to arrest at G1/S in response to checkpoint signals, this information has not led to effective strategies to treat RB1-deficient tumors, and it has been challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/p53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, and we confirmed this was the mechanism of protection in the RP-Skp2 KO mice by using the knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind to and/or inhibit Skp2 induced apoptosis and inhibited SCLC cell growth. In a panel of SCLC cell lines, growth inhibition by a Skp2 inhibitor was not correlated with sensitivity/resistance to etoposide. Targeting Skp2 also had in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using the genetic and pharmacologic approaches, antitumor activity was seen in vivo in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. The identification and validation of an actionable target downstream of RB1 could have a broad impact on treatment of SCLC and other advanced tumors with mutant RB1, for which there are currently no targeted therapies available. Citation Format: Hongling Zhao, Vineeth Sukrithan, Niloy Iqbal, Cari Nicholas, Yingjiao Xue, Joseph Locker, Juntao Zou, Liang Zhu, Edward L. Schwartz. Genetic and pharmacologic inhibition of Skp2, an E3 ubiquitin ligase and RB1-target, has antitumor activity in RB1-deficient human and mouse small cell lung cancer (SCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1060.

Targeted Inhibition of the E3 Ligase SCFSkp2/Cks1 Has Antitumor Activity in RB1-Deficient Human and Mouse Small-Cell Lung Cancer.

The RB1 tumor suppressor gene is mutated in highly aggressive tumors including small-cell lung cancer (SCLC), where its loss, along with TP53, is required and sufficient for tumorigenesis. While RB1-mutant cells fail to arrest at G1-S in response to cell-cycle restriction point signals, this information has not led to effective strategies to treat RB1-deficient tumors, as it is challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, which was confirmed as the mechanism of protection by using knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind/inhibit Skp2 have in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using genetic and pharmacologic approaches, antitumor activity was seen with Skp2 loss or inhibition in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. Our data highlight a downstream actionable target in RB1-deficient cancers, for which there are currently no targeted therapies available. SIGNIFICANCE: There are no effective therapies for SCLC. The identification of an actionable target downstream of RB1, inactivated in SCLC and other advanced tumors, could have a broad impact on its treatment.

Abstract 1306: SEL120, a potent and specific inhibitor of CDK8 induces complete remission in human patient derived xenograft models of acute myeloid leukemia

Abstract Cyclin-dependent kinase 8 inhibitors (CDK8i) have anti-cancer activity in human acute myeloid leukaemia (AML) cell lines both in vitro and in vivo. Activity of CDK8i often involves deregulation of super-enhancer-associated genes in AML cell lines. Previous studies established SEL120 as a specific CDK8 inhibitor active in AML cells with increased STAT1/5 signalling pathways. Differential gene expression analysis demonstrated high enrichment of leukaemia stem cell (LSC) signatures in responding cells, linked to resistance to standard therapies and relapsed disease. Cells sensitive to SEL120 treatment were positive for CD34 and negative for lineage commitment surface markers. SEL120 markedly reduced STAT5 phosphorylation on serine 726 (STAT5 pS726) in sensitive cell lines. Prolonged SEL120 treatment led to significant downregulation of CD34 and induction of lineage commitment markers. Transcriptomic analysis revealed that SEL120 regulated many genes involved in differentiation and apoptosis. We observed synergistic effects of SEL120 with standard of care cytotoxic drugs such as cytarabine. Treatment of AML cells with cytarabine spared many CD34+ cells, which could be effectively eradicated by subsequent treatment with SEL120. Many cell lines which were resistant to SEL120 treatment could be sensitized by concomitant treatment with BH3 mimetic agent ABT-199. Combination of both compounds resulted in potent induction of apoptosis in AML cells in vitro and in vivo. Treatment of mice bearing subcutaneously implanted human leukaemia cell lines resulted in significant tumour growth inhibition, whereas cotreatment with ABT-199 led to complete regressions at doses which were vey well tolerated by animals. Next we have selected patient derived primary AML cells using gene expression signatures identifying SEL120 -responder cell lines. In these cells SEL120 significantly reduced viability, induced apoptosis and lineage commitment. Further, the same cells were implanted into NOD scid gamma mice. Animals succumbed to AML, diagnosed by a significant presence of human CD45/CD34 positive leukaemia cells in a peripheral blood and splenomegaly. Stand-alone treatment with SEL120 resulted in the complete remission of AML cells in a peripheral blood and bone marrow, and reduced spleen weight, without symptoms of compound-related toxicity. These results validate SEL120 as a promising agent in the treatment of AML. Citation Format: Milena Mazan, Eliza Majewska, Michal Mikula, Katarzyna Wiklik, Michal Combik, Aniela Golas, Magdalena Masiejczyk, Elzbieta Fiedor, Anna Polak, Magdalena Cybulska, Aleksandra Grochowska, Michal Kopczynski, Urszula Kuklinska, Zuzanna Sandowska-Markiewicz, Malgorzata Statkiewicz, Agnieszka Paziewska, Michalina Dabrowska, Arkadiusz Bialas, Maciej Mikulski, Renata Windak, Jerzy Ostrowski, Przemyslaw Juszczynski, Krzysztof Brzozka, Tomasz Rzymski. SEL120, a potent and specific inhibitor of CDK8 induces complete remission in human patient derived xenograft models of acute myeloid leukemia [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 1306.

Abstract 4759: Antitumor activity of eribulin after fulvestrant plus palbociclib treatments in the HR(+) human breast PDx model

Abstract Fulvestrant (FLV) and Palbociclib (Palb), which have an effect on degradation of estrogen receptor (ER) and inhibition of cyclin dependent kinases 4 and 6 (CDK4/6), respectively are used for treatments to postmenopausal breast cancer patients. However, there is not a clinical rationale to define a standard treatment regimen against breast cancer patients who are resistant to endocrine therapy plus Palb. Eribulin is a fully synthetic analog of the marine sponge natural product halichondrin B. Its clinical formulation is currently approved for advanced breast cancer (BC) and advanced liposarcoma in numerous countries. We examined the antitumor activity of eribulin after endocrine therapy (combination of FLV and Palb) compared with that of Capecitabine (Cape) in the luminal type breast cancer PDx model (OD-BRE-0438). OD-BRE-0438 is a patient derived xenograft (PDx) model developed from patient with luminal B breast cancer. Gene expression analysis by RNA-Seq showed that OD-BRE-0438 highly expressed ER and PR, but not ERBB2. OD-BRE-0438 tumor bearing mice were treated for 2 weeks of FLV (5mg/ head, Q7Dx2, sc) and Palb (10mg/kg, Q1Dx14, po), and treated mice were randomly divided using tumor volume around 350 mm3 as an index (day1). After that, mice were treated with eribulin (0.5 or 1 mg/kg, which are 1/4 MTD or 1/2 MTD in this model, Q7Dx2, iv) or Cape (125 or 250 mg/kg, which are 1/2 MTD or MTD in this model, Q1Dx14, po for 2 weeks). We measured tumor volume of each mouse after treatments (day 15) and analyzed the ratio of tumor volume at day 15 divided by that at day 1 (RTV: relative tumor volume). Treatments of FLV and Palb nearly completely inhibited in vivo growth of OD-BRE-0438 tumors, but didn’t cause tumor shrinkage. After two weeks of combination treatment with endocrine therapy, we started treatments with either eribulin or Cape, and compared the antitumor activity. Both drug showed strong antitumor activity with tumor shrinkage. In comparison of antitumor activity with either eribulin or Cape, that of eribulin was significantly greater than Cape. [eribulin 0.5mg/kg (RTV=0.42) vs Cape 125mg/kg (RTV=0.62, p=0.0068), eribulin 1.0mg/kg (RTV=0.32) vs Cape 250mg/kg (RTV=0.59, p=0.0008)]. Further investigation for mechanism of action of eribulin to show clear antitumor activity after treatments of FLV and Palb in the luminal type breast cancer PDx model are warranted. Citation Format: Makoto Asano, Takayuki Nakagawa, Taro Semba, Akira Yokoi, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of eribulin after fulvestrant plus palbociclib treatments in the HR(+) human breast PDx model [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 4759.

Abstract 4708: Investigating triple negative breast cancer phenotypic heterogeneity of human and patient-derived xenograft samples using imaging mass cytometry

Abstract Tumors are highly heterogeneous populations of cells, and measures of intratumoral heterogeneity (ITH) and diversity correlate with worse prognosis in many cancers, including breast cancer. Emerging studies are highlighting functional interactions between subclones, as well as among subclones and components of the tumor microenvironment. However, these studies have largely focused on soluble factors without interrogating the spatial distribution of subclones defined by activated signaling pathways. Previous work in this area has been severely limited by technical restrictions - existing techniques allowing measurement of many biomarkers simultaneously lose all information about the tissue architecture, while those that do retain spatial information can only assay a handful of markers at once. We will circumvent these limitations by undertaking imaging mass cytometry (IMC), which allows for simultaneous measurement of 30-40 antigens while retaining the spatial organization of the sample. Our objective is to dissect the signaling heterogeneity in tumors from patients and patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), through two main approaches: (1) characterization of signaling heterogeneity in tissue microarrays of human tumors and PDX models of TNBC with IMC and (2) modeling cell signaling heterogeneity in cell line-based models to determine mechanisms of cell-cell interaction and communication. We have constructed an IMC panel of antibodies that combines markers for tissue architecture, tumor and immune cell phenotyping, and signaling pathway activation. Profiling of a diverse panel of TNBC PDX models captures the heterogeneity of human TNBC. Analysis of distinct regions within individual PDX tumors demonstrate unique compositions of cell phenotypes between the edge and core of the tumor. Our PDX collection also includes sequential pairs derived from biopsies taken before and after chemotherapy treatment. Comparison of the pre-/post-chemotherapy pairs indicates emerging patterns of pathway activation. Interestingly, while the tumor cells from these models exhibit distinct phenotypes, the stromal cells are largely indistinguishable from one another, suggesting that these models are capturing tumor cell-intrinsic changes associated with chemotherapy response. Ultimately, we plan to use these results to identify novel vulnerabilities in subclonal interactions that can be targeted therapeutically in TNBC. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Xiaomei Zhang, Jiansu Shao, Helen Piwnica-Worms. Investigating triple negative breast cancer phenotypic heterogeneity of human and patient-derived xenograft samples using imaging mass cytometry [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 4708.

Irreversible electroporation stimulates a pro-inflammatory tumor microenvironment in pancreatic cancer

Abstract Pancreatic cancer is a major cancer with a survival rate of only 6% due to late diagnosis and the aggressiveness of the malignancy. There is also a severe lack of viable treatment options due to the localization of the primary tumor near major blood vessels and bile ducts as well as the highly immunosuppressive nature and metastatic potential of pancreatic cancer. New ablative techniques for pancreatic tumors are being developed; however most are thermal and can cause damage to the delicate surrounding structures. Non-thermal irreversible electroporation (IRE) uses short, high frequency electrical pulses to permeabilize cancer cell membranes and illicit cell death. IRE has shown promising results in clinical trials of late-stage, locally advanced cases. Here we utilize several models of pancreatic cancer in vitro, ex vivo, and in vivo to determine the effect of IRE on cell death pathways, cancer pathways, and immune signaling. IRE elicits pro-inflammatory cell death via necrosis and pyroptosis in immunocompetent murine and human patient-derived xenograft models. This pro-inflammatory environment can stimulate immune cell infiltration to the tumor site, halting tumor progression. Moreover, a non-thermal ablative approach also preserves cancer cell antigens that can lead to better immunosurveillance against reoccurrence and metastatic lesions. With the advent of these new techniques, the impact of such technologies to the tumor microenvironment and immune system need to be assessed to determine their clinical application. IRE is proving to be a safe and effective treatment for pancreatic cancer as it not only ablates the primary tumor site but also stimulates the immune system to recognize and combat the tumor throughout the body.

Dual Inhibition of Bcl-2/Bcl-xL and XPO1 is synthetically lethal in glioblastoma model systems

XPO1 has recently emerged as a viable treatment target for solid malignancies, including glioblastoma (GBM), the most common primary malignant brain tumor in adults. However, given that tumors become commonly resistant to single treatments, the identification of combination therapies is critical. Therefore, we tested the hypothesis that inhibition of anti-apoptotic Bcl-2 family members and XPO1 are synthetically lethal. To this purpose, two clinically validated drug compounds, the BH3-mimetic, ABT263, and the XPO1 inhibitor, Selinexor, were used in preclinical GBM model systems. Our results show that inhibition of XPO1 reduces cellular viability in glioblastoma cell cultures. Moreover, addition of ABT263 significantly enhances the efficacy of XPO1 inhibition on the reduction of cellular viability, which occurs in a synergistic manner. While selinexor inhibits the proliferation of glioblastoma cells, the combination treatment of ABT263 and selinexor results in substantial induction of cell death, which is accompanied by activation of effector- initiator caspases and cleavage of PARP. Mechanistically we find that XPO1 inhibition results in down-regulation of anti-apoptotic Mcl-1 and attenuates ABT263 driven Mcl-1 up-regulation. Consistently, siRNA mediated silencing of Mcl-1 sensitizes for ABT263 mediated cell death and partially for the combination treatment. By using a human patient-derived xenograft model of glioblastoma in mice, we demonstrate that the combination treatment of ABT263 and Selinexor reduces tumor growth significantly more than each compound alone. Collectively, these results suggest that inhibition of XPO1 and Bcl-2/Bcl-xL might be a potential strategy for the treatment of malignant glial tumors.

Dual compartmental targeting of cell cycle and angiogenic kinases in colorectal cancer models.

Cancer is a disease caused by several factors characterized by uncontrolled cell division, growth, and survival. ENMD-2076, is a novel orally active small molecule multikinase inhibitor targeting angiogenesis, proliferation, and the cell cycle. It is selectively active against the mitotic kinases aurora A and B, and kinases responsible for angiogenesis including VEGFR2/KDR and FGFR1 and 2. ENMD-2076 has been shown to inhibit tumor growth and prevent angiogenesis in vitro and in vivo in preclinical cancer models. Moreover, in a phase I trial, ENMD-2076 was well tolerated, exhibited a linear pharmacokinetic profile, and showed a promising antitumor activity in a number of solid tumors. In this study, we show that ENMD-2076 has antiproliferative effects, causes cell cycle arrest, and has activity in preclinical models of colorectal cancer (CRC), including patient-derived xenograft (PDX) models. Forty-seven human CRC cell lines were exposed in vitro to ENMD-2076 and analyzed for effects on cell cycle, apoptosis, and downstream effector proteins. The drug was then tested against 20 human CRC PDX models to further evaluate in-vivo antitumor activity. We show that ENMD-2076 exhibits a broad range of activity against a large panel of CRC cell lines with varying molecular characteristics. Mechanistically, ENMD-2076 exposure resulted in a G2/M cell cycle arrest, an increase in aneuploidy, and cell death in responsive cell lines. In addition, ENMD-2076 treatment resulted in a promising antitumor activity in CRC PDX models. These results support the continued development of ENMD-2076 in CRC including further exploration of rational combinations.

Abstract 2181: Functionalizing intratumoral signaling heterogeneity in triple negative breast cancer

Abstract Tumors are highly heterogeneous populations of cells, and measures of intratumoral heterogeneity (ITH) and diversity correlate with worse prognosis in many cancers, including breast cancer. Emerging studies are highlighting functional interactions between subclones, as well as among subclones and components of the tumor microenvironment. However, these studies have largely focused on soluble factors without interrogating the spatial distribution of subclones defined by activated signaling pathways. Previous work in this area has been severely limited by technical restrictions – existing techniques allowing measurement of many biomarkers simultaneously lose all information about the tissue architecture, while those that do retain spatial information can only assay a handful of markers at once. We will circumvent these limitations by undertaking imaging mass cytometry (IMC), which allows for simultaneous measurement of 30-40 antigens while retaining the spatial organization of the sample. Our objective is to dissect the signaling heterogeneity in tumors from patients and patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), through two main approaches: (1) characterization of signaling heterogeneity in human tumors and PDX models of TNBC through the use of imaging mass cytometry (IMC) and (2) modeling cell signaling heterogeneity in cell line-based models to determine mechanisms of cell-cell interaction and communication. We have constructed an IMC panel of antibodies that combines markers for tissue architecture, cell phenotyping, and signaling pathway activation. Profiling of a panel of TNBC PDX models, including matched sets of pre- and post-chemotherapy treatment, indicates emerging patterns of pathway activation. We will utilize these results to inform functional studies in our isogenic cell line models. As it is becoming more clear that tumors are composed of heterogeneous subclones, it is crucial that we understand the communication between subclones and with components of the microenvironment. Ultimately, we plan to use these results to identify novel vulnerabilities in subclonal interactions that can be targeted therapeutically in TNBC. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Xiaomei Zhang, Helen Piwnica-Worms. Functionalizing intratumoral signaling heterogeneity in triple negative breast cancer [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 2181.