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H3B-5942 exhibits dose-dependent inhibition of ER target genes and shows significant efficacy ER Wt and mutant in vivo models

Supplementary materials and methods and supplementary figure legends

Supplementary Table 2 from A Pilot Clinical Study of Treatment Guided by Personalized Tumorgrafts in Patients with Advanced Cancer

In SW48 colorectal cells engineered with KRAS alleles, response to paclitaxel was unaltered.

Supplementary Figure from Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer

Supplementary Figure 1 from A Pilot Clinical Study of Treatment Guided by Personalized Tumorgrafts in Patients with Advanced Cancer

Patients with many advanced solid cancers have very poor prognosis, and improvements in life expectancy are measured only in months. We have recently reported the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA-damaging agents, on the basis of the observation of significant activity of this class of drugs against a personalized tumorgraft generated from the patient's surgically resected tumor. Here, we extend the approach to patients with other advanced cancers. Tumors resected from 14 patients with refractory advanced cancers were propagated in immunodeficient mice and treated with 63 drugs in 232 treatment regimens. An effective treatment regimen in the xenograft model was identified for 12 patients. One patient died before receiving treatment, and the remaining 11 patients received 17 prospectively guided treatments. Fifteen of these treatments resulted in durable partial remissions. In 2 subjects, no effective treatments were found. Overall, there was a remarkable correlation between drug activity in the model and clinical outcome, both in terms of resistance and sensitivity. The data support the use of the personalized tumorgraft model as a powerful investigational platform for therapeutic decision making and to efficiently guide cancer treatment in the clinic. Mol Cancer Ther; 10(8); 1311–6. ©2011 AACR.

Supplementary Data from Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer

Enriched GSEA pathways

Supplementary Table 1 from A Pilot Clinical Study of Treatment Guided by Personalized Tumorgrafts in Patients with Advanced Cancer

Supplementary Figure Legend

Purpose:Antibody–drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance.Experimental Design:The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro. The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3. SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR.Results:Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3, suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs.Conclusions:Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.

Purpose: This study evaluated the clinical relevance of the dual-targeting strategy involving PI3K/AKT/mTOR and RAF/MEK/ERK pathways.Experimental Design: We investigated safety, efficacy, and correlations between tumor genetic alterations and clinical benefit in 236 patients with advanced cancers treated with phase I study drugs targeting phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways in our Phase I Clinical Trials Program.Results: Seventy-six (32.2%) patients received a PI3K pathway inhibitor in combination with a MAPK pathway inhibitor (D), whereas 124 (52.5%) and 36 (15.3%), respectively, received an inhibitor of either the PI3K or MAPK pathways (S). The rates of drug-related grade >III adverse events were 18.1% for (S) and 53.9% for (D; P < 0.001); the rates of dose-limiting toxicities were 9.4% for (S) and 18.4% for (D; P = 0.06). The most frequent grade >III adverse events were transaminase elevations, skin rash, and mucositis. In our comprehensive tumor genomic analysis, of 9 patients who harbored coactivation of both pathways (colorectal cancer, n = 7; melanoma, n = 2), all 5 patients treated with (D) had tumor regression ranging from 2% to 64%.Conclusions: These results suggest that dual inhibition of both pathways may potentially exhibit favorable efficacy compared with inhibition of either pathway, at the expense of greater toxicity. Furthermore, this parallel pathway targeting strategy may be especially important in patients with coexisting PI3K pathway genetic alterations and KRAS or BRAF mutations and suggests that molecular profiling and matching patients with combinations of these targeted drugs will need to be investigated in depth. Clin Cancer Res; 18(8); 2316–25. ©2012 AACR.

CCR Translation for This Article from The Clinical Effect of the Dual-Targeting Strategy Involving PI3K/AKT/mTOR and RAS/MEK/ERK Pathways in Patients with Advanced Cancer

Purpose: There are no effective treatment options for patients with advanced adenoid cystic carcinoma (ACC). Here, we evaluated the effect of a new small molecule inhibitor of the MDM2–p53 interaction (MI-773) in preclinical models of ACC.Experimental Design: To evaluate the anti-tumor effect of MI-773, we administered it to mice harboring three different patient-derived xenograft (PDX) models of ACC expressing functional p53. The effect of MI-773 on MDM2, p53, phospho-p53, and p21 was examined by Western blots in 5 low passage primary human ACC cell lines and in MI-773-treated PDX tumors.Results: Single-agent MI-773 caused tumor regression in the 3 PDX models of ACC studied here. For example, we observed a tumor growth inhibition index of 127% in UM-PDX-HACC-5 tumors that was associated with an increase in the fraction of apoptotic cells (P = 0.015). The number of p53-positive cells was increased in MI-773-treated PDX tumors (P < 0.001), with a correspondent shift in p53 localization from the nucleus to the cytoplasm. Western blots demonstrated that MI-773 potently induced expression of p53 and its downstream targets p21, MDM2, and induced phosphorylation of p53 (serine 392) in low passage primary human ACC cells. Notably, MI-773 induced a dose-dependent increase in the fraction of apoptotic ACC cells and in the fraction of cells in the G1 phase of cell cycle (P < 0.05).Conclusions: Collectively, these data demonstrate that therapeutic inhibition of the MDM2–p53 interaction with MI-773 activates downstream effectors of apoptosis and causes robust tumor regression in preclinical models of ACC. Clin Cancer Res; 22(14); 3550–9. ©2016 AACR.

Supplementary methods and materials, and Supplemental Figures

Table S1: (Table depicting patient demographics and tumor characteristics); Table S2: (Table depicting STR profiles of experimental models used here); Figure S1: (Characterization of UM-PDX-HACC-5, a PDX model of adenoid cystic carcinoma); Figure S2: (MI-773 induces ACC tumor regression); Figure S3: (MI-773 induces ACC tumor regression in an additional model of ACC); Figure S4: (Effect of MI-773 is p53-dependent); Figure S5: (Histopathology of UM-PDX-HACC-5 tumors treated with MI-773); Figure S6: (Aminoacid sequence of p53 in the ACC models used in this project)

Supplementary material Supplementary Table 1. Customized primer panel for Sequenom MassARRAY® somatic mutation profiling of DNA from models of adenoid cystic carcinoma. Supplementary Figure 1. Apparent oral clearance (CL/F) of dacomitinib 10 mg (D-1b) and 15 mg (D-1d) in the presence of figitumumab (20 mg/kg). Boxes, 25th-75th percentiles (1st-3rd quartiles); circles individual patient values; horizontal black bars, medians; whiskers, 1.5 Ã- interquartile range. Supplementary Figure 2. Dose-normalized maximal plasma concentration (Cmax; A) and area under the concentration-time curve (AUC24; B) for dacomitinib 10 mg (D-1b) and 15 mg (D-1d) in the presence of figitumumab (20 mg/kg) (Pfizer, data on file). Boxes, 25th-75th percentiles (1st-3rd quartiles); circles individual patient values; horizontal black bars, medians; whiskers, 1.5 Ã- interquartile range. Supplementary Figure 3. Apparent oral clearance (CL/F) of dacomitinib in the presence (cohorts D-1b and D-1d in this study [study 1004]) and the absence (studies 1001, 1003, and 1005) of figitumumab (20 mg/kg) (Pfizer, data on file). Boxes, 25th-75th percentiles (1st-3rd quartiles); circles individual patient values; horizontal black bars, medians; whiskers, 1.5 Ã- interquartile range. Supplementary Figure 4. Dose-normalized maximal plasma concentration (Cmax; A) and area under the concentration-time curve (AUC24; B) for dacomitinib in the presence (cohorts D-1b and D-1d in this study [study 1004]) and the absence (studies 1001, 1003, and 1005) of figitumumab (20 mg/kg) (Pfizer, data on file). Boxes, 25th-75th percentiles (1st-3rd quartiles); circles individual patient values; horizontal black bars, medians; whiskers, 1.5 Ã- interquartile range.

Supplementary table showing the relative expression of 43 target genes in a panel of EphA2-positive cell lines.

Purpose: This phase I, open-label, single-arm trial assessed the safety and tolerability of dacomitinib–figitumumab combination therapy in patients with advanced solid tumors.Experimental Design: A standard 3 + 3 dose escalation/de-escalation design was utilized. Starting doses were figitumumab 20 mg/kg administered intravenously once every 3 weeks and dacomitinib 30 mg administered orally once daily. We also performed an independent study of the combination in patient-derived xenograft (avatar mouse) models of adenoid cystic carcinoma.Results: Of the 74 patients enrolled, the most common malignancies were non–small cell lung cancer (24.3%) and colorectal cancer (14.9%). The most common treatment-related adverse events in the 71 patients who received treatment across five dose levels were diarrhea (59.2%), mucosal inflammation (47.9%), and fatigue and acneiform dermatitis (45.1% each). The most common dose-limiting toxicity was mucosal inflammation. Dosing schedules of dacomitinib 10 or 15 mg daily plus figitumumab 20 mg/kg every 3 weeks after a figitumumab loading dose were tolerated by patients over multiple cycles and considered recommended doses for further evaluation. Objective responses were seen in patients with adenoid cystic carcinoma, ovarian carcinoma, and salivary gland cancer. Pharmacokinetic analysis did not show any significant drug−drug interaction. In the adenoid cystic carcinoma xenograft model, figitumumab exerted significant antitumor activity, whereas dacomitinib did not. Figitumumab-sensitive tumors showed downregulation of genes in the insulin-like growth factor receptor 1 pathway.Conclusions: Dacomitinib−figitumumab combination therapy was tolerable with significant dose reductions of both agents to less than the recommended single-agent phase II dose of each drug. Preliminary clinical activity was demonstrated in the potential target tumor adenoid cystic carcinoma. Clin Cancer Res; 23(5); 1177–85. ©2016 AACR.See related commentary by Sundar et al., p. 1123

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy–resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). Summary: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Background: Mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) have been well studied and several alterations identified including RB loss and altered expression of related genes including CCNE1, E2F4 and CDK6. However, whether duration of clinical treatment might elicit specific mechanism(s) of CDK4/6i resistance is unclear. To better understand if duration of clinical treatment correlates with unique resistance mechanisms, we established, characterized, and compared a panel of ER+ breast XPDX models from patients who benefitted then progressed on a CDK4/6i. Patients were separated into two groups by time to progression (TTP): those who responded up to twelve months (RES12) and patients with clinical response greater than one year (RES13+). Methods: Seventeen breast cancer XPDX models were analyzed, including eight previously described (7xRES12; 1xRES13+: SABCS2021: T Hernandez et al). Nine new models were established from seven patients: six from fluid samples with three designated as ductal (ST3105B, ST3105C, STM001B) and three lobular carcinoma (STM182, STM229, STM229B); two from lymph node biopsies (ST5676, STM127) and one from a liver core biopsy (ST4887B), all reported as ductal carcinoma. STM182 was classified as RES12 and the remaining eight as RES13+. These models were passaged and challenged with CDK4/6i to confirm resistance. Receptor expression was determined by IHC and genomic analyses including WES and RNAseq, were performed to identify mechanisms of resistance. For in vivo studies, CDK4/6i were dosed PO once daily at 50 mg/kg; endpoints included tumor volume (TV) and time from treatment initiation (TTI) with %T/C values and tumor regression reported at study completion; a %T/C of ≤20 versus control was considered sensitive. Tumor regression (%T/C Results: Clinical TTP for RES12 (n=8) was four to twelve months and RES13+ (n=9) from thirteen to forty-two months. All models retained ER expression in evaluated passages with similar histology compared with archival clinical samples. Sequencing identified several variants including RB1 truncations or deletions and increased gene expression in CCND1, CCNE1 and the PIK3CA/AKT pathway. Interestingly, 5/8 RES12 models reported ESR1 mutations or fusions versus 1/9 RES13+ and PIK3CA mutations were reported in 1/8 RES12 versus 5/9 RES13+. Several RES13+ models also reported variants and increased amplification in the RICTOR/TORC2 pathway versus RES12. Conclusion: We have established, characterized, and compared a panel of seventeen breast XPDX models from fourteen female patients representing early or late acquired resistance to CDK4/6i therapy and identified potential differences in each set. These models and resulting data are useful in developing novel therapies for CDK4/6i-resistant patients. Citation Format: Alyssa Simonson, Johnnie Flores, Morgan Lynch, Emily Carpenter, Justine Hruzek, Jim Lund, Natalia Baños Herraiz, Kyriakos Papadopoulos, Amy Vander Woude, Gladys Rodriguez, Sreenivasa Chandana, Thomas Gribbin, Nehal Lakhani, Tatiana Hernandez, Maria Jose de Miguel, Amy Lang, Michael J. Wick. Short or long-term treatment with CDK4/6 inhibitors in patients with ER+ breast cancer: characterization and comparative analysis of resistance in seventeen XPDX 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 3869.

Biological therapies for the treatment of cancer are utilized for the same ultimate purpose as chemically derived cancer drugs, to induce tumor cell death. However, the differing mechanisms of action often make the evaluation of biologic drug efficacy more complex, requiring the support and/or recapitulation of functional immune components. Biologics also have different limitations that can govern their efficacy such as their large size potentially impeding sufficient penetration to target sites and in the case of live biotherapeutic products, the need to maintain sustained function, or persistence, by resisting immunosuppression by the tumor microenvironment. Preclinical testing of biologics is often confined to cell lines which lack an immune system and mouse models which are expensive, time consuming, and poor proxies for the human immune system. To address these issues, we have developed ex vivo 3D spheroid platforms to measure the efficacy of three types of biologics: antibody-drug conjugates (ADCs), bispecific antibodies, and chimeric antigen receptor (CAR) T-cells. Primary cells from cancer patients or from patient-derived xenografts were evaluated for the expression of the biologic target molecule then cultured to form 3D spheroids. Spheroid models were validated for the evaluation of antibody-based therapies by determining sufficient diffusion of antibodies to target sites. For ADC testing, cell death was measured in a plate-based read-out following treatment with the ADC trastuzumab deruxtecan, and the cytotoxic efficacy of the ADC was compared to trastuzumab alone or free exatecan. Differences in killing kinetics and the maximum efficacy of the ADC were determined compared to the other tested agents. HER2 inhibition was evaluated through changes in constitutive downstream signaling. Two biologics with the same molecular target, a bispecific antibody targeting carcinoembryonic antigen (CEA) on tumor cells and CD3 on T-cells, as well as a CAR T-cell product targeting CEA were also evaluated. Following treatment with the bispecific, tumor cell viability was detected using flow cytometry and changes in T-cell activation were measured via cytokine secretion using primary colorectal cancer models. Importantly, bispecific antibody efficacy was dependent upon the presence of T-cells. Finally, CAR T-cell mediated tumor cell killing was detected using a fluorescent image-based approach. Collectively, this work establishes a model for testing multiple classes of drugs in a 3D ex vivo model that can be used to advance preclinical drug development and evaluate mechanisms of action of diverse drug classes in a physiologically relevant model. Citation Format: Katy A. Lassahn, Ashley K. Elrod, Aaron L. Carlson, Natalie A. Dance, Melissa Millard, Michael J. Wick, Teresa M. DesRochers, Kathryn M. Appleton. Preclinical testing of therapeutic biologics using patient-derived 3D spheroids [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 2275.

Prior to entering clinical testing, a new molecular entity must first undergo a series of rigorous scientific assessments. These include pharmacologic evaluations of safety and efficacy, toxicology testing under carefully controlled conditions, and the thorough characterization of manufacturing processes. Nonclinical development, which spans the gap between drug discovery and clinical testing, encompasses all of these activities. The data resulting from these evaluations are subject to rigorous regulatory review prior to the initiation of first-in-human (FIH) trials. This chapter describes the general process of nonclinical drug development, including the requirements for an FDA IND filing with specific examples taken from the field of oncology.

Background: Palbociclib is one of three CDK 4/6 inhibitors approved in combination with an aromatase inhibitor for treatment of hormone receptor-positive breast cancer. Although this combination therapy has been found effective in some patients, resistance often develops. To aid in developing new therapies for CDK4/6-resistant breast cancer and better understand resistance mechanisms, we established a PDX model, designated ST1799, from a patient with luminal A breast cancer which was responsive to palbociclib in vivo and then conditioned the model with chronic drug administration until resistant. The resistant model, designated ST1799/PBR, and the parent were characterized for receptor expression, genomic alterations and in vivo drug sensitivity. Methods: ST1799 was established from an axillary lymph node FNA taken from a 66-year-old woman pretreated with various chemo and hormone therapies. The resulting model was passaged and a cohort challenged with chronic palbociclib treatment to produce drug resistance. The parent ST1799 and resistant ST1799/PBR were subjected to various comparative analyses including receptor expression, NGS and RNAseq and response to various relevant therapies. Results: Both models retained high ER staining (3+) expression and demonstrated comparable histopathology. DNA-based analysis of the parent model confirmed variants concordant with clinical mutation analysis. Comparison of the parent and resistant clone using RNAseq identified several alterations in variants and expression. In vivo palbociclib reported significant (p Citation Format: Lizette Gamez, Alyssa Moriarty, Tomoyuki Mashimo, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Michael J Wick. Establishment and characterization of paired palbociclib-sensitive and resistant luminal A breast PDX models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-10.

Background: Preclinical models of prostate cancer are challenging to develop and maintain, especially those that grow in a castrated setting while maintaining receptor and antigen expression. To this end, we have established and characterized a panel of XenoSTART patient-derived (XPDX) prostate models using both intact and castrated athymic nude mice. These models, designated ST1273, ST2347, ST4017, and ST4420, were characterized for receptor expression, genomic alterations, and in vivo drug sensitivity to relevant therapies. Methods: XPDX models representing prostate cancer were established from primary (ST1273) or metastatic (ST2347, ST4017, ST4420) biopsy samples implanted into intact athymic nude mice supplemented with exogenous testosterone. Resulting models were passaged and further developed in both intact and castrated athymic nudes until growth stabilization. Resulting models were characterized using genomic analysis, including WES and RNAseq, receptor expression, and in vivo drug sensitivity studies. Models found sensitive to enzalutamide were conditioned to resistance in vivo by chronic drug administration and resulting models (designated STxxxx/EZR) were characterized and compared with parent lines. For in vivo studies, activity of relevant treatments were benchmarked including enzalutamide administered once daily by oral gavage at 50 mg/kg and docetaxel administered by intravenous injection once weekly at 10 mg/kg. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Results: Each model developed in a castrate or conditioned setting retained similar receptor expression to the parent model including positive AR (2+/3+) and PSMA (2+/3+) staining. Genomic characterization identified a PIK3CA mutation in ST1273 models (PIK3CAE545A), AR mutations in ST2347 (ART878A) and ST4017 (ARH875Y), and a TMPRSS2:ERG fusion in ST4420. In vivo, the ST1273 parent model was found sensitive to enzalutamide (%T/C=11%) but insensitive in castrated mice (%T/C=76%) and the ST1273/EZR model was resistant to enzalutamide in intact (%T/C=56%) or castrated (%T/C=100%) mice. The ST2347 parent model was also found sensitive to enzalutamide (%T/C=19%) but insensitive in castrated mice (%T/C=50%). ST4017 and ST4420 studies in intact and castrated mice are currently underway. All models were sensitive to docetaxel. Conclusion: We have established and characterized a panel of prostate XPDX models using both intact and castrated athymic nude mice and conditioned resistance to enzalutamide by chronic drug administration. These models can be utilized as a valuable tool in better understanding castrate-resistant prostate cancer and in developing novel therapies for enzalutamide-resistant patients. Citation Format: Johnnie Flores, Alyssa Simonson, Dustin Kneifel, Alejandra Diaz, Morgan Harris, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Scott Ulmer, Michael J. Wick. Establishment and characterization of a panel of castrate-resistant prostate cancer XPDX models with differential enzalutamide response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3109.

Background: First-line treatment for some cancer types includes platinum-based therapy. While the rate of initial response to treatment is high, most patients develop platinum-resistant disease. Current salvage therapy provides benefit to some patients, demonstrating the need for additional effective therapies. To assist in identifying new therapies, we have established XenoSTART patient-derived xenograft (XPDX) models representing breast, ovary, uterine, and lung cancers. Each model was characterized by DNA/RNA analysis, sensitivity to platinum treatment, and annotated with donor patient treatment status at the time of sample collection. Methods: 210 XPDX models were evaluated in this screen including 66 breast, 56 ovary, 36 uterine, and 52 lung xenografts. WES and RNAseq were performed on each model and patient treatment history and outcome annotated. For in vivo studies, models were implanted into female nudes and administered (IP; q7dx3) 3 m/k cisplatin or 60 m/k carboplatin. Study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Results: Study results are summarized below in Table 1: Sequencing identified several variants in resistant, chemo-naïve models including point mutations in RAS/RAF/MET/PIK3CA genes and others, while sensitive models lacked common driver mutations. Conclusion: We have characterized 210 XPDX models. Uterine models were most often resistant to platinum, while almost 1/2 of ovary and 1/3 of breast models were sensitive, regardless of treatment status. Only 15% of lung models were sensitive to platinum; cancer driver variants were found in several models. Overall, we report differential platinum sensitivity in a panel of diverse models useful in understanding mechanisms of resistance and for development of effective therapies in platinum-resistant cancers. Table 1. Type Breast Ovary Uterine Lung Total 66 56 36 52 # Sensitive 25 25 9 8 % 38% 45% 25% 15% Naïve 11 16 8 5 % 44% 64% 89% 63% P-1st 6 6 1 2 % 24% 24% 11% 25% P-2nd+ 6 3 0 1 % 24% 12% 0% 13% # Insensitive 41 31 27 44 % 62% 55% 75% 85% Naïve 11 9 20 21 % 27% 29% 74% 48% P-1st 14 10 5 13 % 34% 32% 19% 30% P-2nd+ 16 12 2 10 % 39% 39% 7% 23% Naïve=No Prior Treatment; P-1st=Post 1st Line Therapy; P-2nd=Post 2nd+ Line Therapy Citation Format: Alyssa Simonson, Johnnie Flores, Lizette Firova, Christian Hernandez, Morgan Harris, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Allan White, Lon Smith, Ronald Drengler, Amy Lang, Murali Beeram, Michael J. Wick. Correlation of platinum sensitivity with donor patient treatment status in a panel of breast, ovary, uterine, and lung XPDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1092.

Patient-Derived Xenografts (PDX) represent a versatile tool for preclinical drug development because they recapitulate many key features of the parent tumors, including molecular and histopathological profiles, tumor microenvironment, and tumor heterogeneity. The clinical relevance of PDX thus offer several advantages over other commonly used tools such as cell lines and genetically modified mice. Many large collections of PDX have been developed across a wide range of tumor types that profile the genetic diversity of each disease and the ability to bank PDX material allows repeated generation of mice for in vivo drug screens. As a result, PDX are used in several parts of the drug development pipeline, including early cancer biology studies, biomarker development, evaluation of therapeutic efficacy, and assessing/overcoming drug resistance. However, there remain drawbacks to this approach, most notably the large number of mice required for comprehensive drug screens and the associated time and costs. One approach to streamline PDX model selection and in vivo study execution would be to predict in vivo drug responses by assessing responses to the same drugs in an ex vivo platform utilizing PDX-derived dissociated tumor cells. KIYATEC’s KIYA-PREDICT™ PDX assay is a 3D spheroid-based ex vivo platform that has been used to screen a wide range of drugs and tumor types to predict drug responses in primary patient-derived tumors and PDX-derived tumors, including several PDX from XenoSTART’s extensive library of XPDX models. Here, we dissociated XPDX-derived tumors from a panel of 20 breast, ovarian, and lung cancer models provided by XenoSTART and evaluated their ex vivo responses to a panel of chemotherapy agents in our 3D KIYA-PREDICT™ assay. After exposure to drugs for 3-7 days, viability was assessed and both IC50s and percent survival values were calculated. The percent survival was then compared to in vivo drug response data provided by XenoSTART, and correlations between in vivo and ex vivo data were assessed. Drug responses were highly correlative between ex vivo and in vivo models, including the ability to recapitulate palbociclib resistance and platinum and taxane response. These results indicate that the KIYA-PREDICT™ PDX assay is a valuable tool to incorporate into drug development pipelines to accelerate the screening of new drug compounds on a wide range of clinically relevant samples and to guide selection of PDX models for in vivo studies. Citation Format: Aaron L. Carlson, Ashley K. Elrod, Natalie A. Williams, Alyssa D. Moriarty, Michael J. Wick, Teresa M. DesRochers. Ex vivo 3D drug response profiling of XPDX-derived tumor cells for acceleration of preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 183.

Background: Neuregulin-1 (NRG1) rearrangements drive cancers by binding to ERBB3/ERBB2 heterodimers and activating downstream signaling. NRG1 fusion proteins have recently been identified in several cancer types including lung and ovary. To better understand the role of NRG1 fusions in cancer we established three XenoSTART patient-derived xenograft (XPDX) models driven by NRG1 rearrangements including two CD74-NRG1 lung models designated ST2891 and ST3204 and one SARAF-NRG1 ovary model designated ST2476. These models were established in athymic nude mice and characterized for receptor expression, genomic alterations, and in vivo drug sensitivity. Methods: ST2891 was established from an excision biopsy collected from a 68-year-old male with NSCLC following treatment with carboplatin/paclitaxel and carboplatin/pemetrexed. ST3204 was established from a lymph node biopsy collected from a 63-year-old female with NSCLC following treatment with cisplatin/pemetrexed. ST2476 was established from primary tissue collected from a chemo naïve 61-year-old female with ovarian cancer. The resulting models were passaged and characterized using immunohistochemical and genomic analysis including WES and RNAseq. In vivo studies were performed testing various chemotherapy and targeted agents; study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C Results: All models reported ERBB2 and ERBB3 staining in evaluated passages with similar histology compared with archival clinical samples. WES analysis of the three models did not identify any variants in EGFR/ERBB2-4/RAS/RAF genes. RNA sequencing revealed CD74-NRG1 or SARAF-NRG1 gene fusions independently confirmed using molecular studies. In vivo, both lung models were resistant to weekly cisplatin or T-DM1 and daily palbociclib (50 mg/kg) or daily afatinib up to 10 mg/kg. ST3204 was found sensitive to weekly pertuzumab (1 mg/kg). Both lung models were resistant to trastuzumab (1 mg/kg). The ST2476 ovary XPDX was found resistant to endocrine therapies and reported some sensitivity to weekly cisplatin. Conclusion: We have established and characterized a panel of three XPDX models driven by NRG1 rearrangements including two CD74-NRG1 lung models and one SARAF-NRG1 ovary model. These models are valuable tools for further developing therapies targeting NRG1-driven cancers. Citation Format: Alyssa Simonson, Johnnie Flores, Crystal Moreno, Justine Hruzek, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Ronald Drengler, Allan White, Jun Ma, Michael J. Wick. Establishment and characterization of neuregulin-1 (NRG1) fusion driven XPDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 353.

Adenoid cystic carcinoma (ACC) is a rare, glandular cancer whose incidence rate and limited 2D cell culture capability make it difficult to study primary patient samples. Although well characterized ACC patient-derived xenografts (PDX) have proven to be a useful model to study disease mechanisms and therapeutic sensitives, animal drug studies are relatively low throughput, costly, and take months to accomplish. Ex vivo 3D cell culture can provide a high-throughput, less costly, and significantly faster platform for these drug studies but are hampered by the rarity of tissue. To address this unmet need for models of rare tumor types we developed 3D spheroid (3D-XPDXs™) and microtumor (3D-XPDXmt™) models of ACC using PDX as the primary tissue source. ACC PDX cells readily formed spheroids in our 3D-XPDXs™ platform, remained viable for up to 14 days, and maintained disease-relevant biomarkers such as MYB and c-kit. 3D-XPDXmt™ represent a more complex model of ACC by incorporating extracellular matrix. ACC 3D-XPDXmt™ displayed tumor-like morphologies concordant with the parental tumors and exhibited MYB and c-kit biomarker expression for up to 56 days in culture. Drug response profiling (DRP) was performed using the 3D-XPDXs™ ACC model with KIYATEC’s validated KIYA-PREDICT™ DRP platform. The screening panel consisted of drugs and drug-like compounds currently in use or under investigation for use in ACC, including broad-spectrum chemotherapies and targeted agents. Individualized drug responses were noted for each model as they exhibited differential sensitivities to DNA-damaging agents, microtubule stabilizers, and c-kit targeting kinase inhibitors mirroring the diversity of clinical outcomes. KIYA-PREDICT™ also identified drugs and drug-like compounds that uniformly inhibited viability. This is significant because there is currently no standard of care drugs for ACC, as few, if any have demonstrated homogenous responses in test populations. Monensin has been shown to inhibit activity of the MYB transcription factor, making it an attractive candidate drug for the treatment of ACCs which have a high prevalence of MYB activating alterations. In our study, monensin was effective in all three models with IC50 concentrations in the low micromolar range. These results correlate well with immunohistochemical staining of MYB in ACC 3D-XPDXs™ and 3D-XPDXmt™. Taken together, this data represents a new ex vivo 3D cell culture platform for the study of ACC biology and potential therapies. Citation Format: Melissa Millard, Teresa M. DesRochers, Michael J. Wick, Jeffrey Kaufman, Nicole Spardy Burr. Ex vivo 3D culture of adenoid cystic carcinoma PDX models recapitulate disease biomarkers and predict drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 177.

Background: We previously reported (RAS AACR 2022) a preclinical screen testing KRASG12C-inhibitors (KRASG12C-i) in 13 colorectal and 13 lung KRASG12C XPDX models; all tested models were from patients naïve to KRASG12C-i. To better understand mechanisms of resistance in this agent class, we established three lung XPDX models designated ST5185B, ST5431B, and ST5489 from patients before therapy with a KRASG12C-i. We also established one colorectal XPDX, designated ST4859, post therapy. Once established these models were molecularly characterized, in vivo sensitivity to sotorasib and adagrasib determined, and results compared with clinical response. Ex vivo cultures and 3D-XPDX࣪ studies were also performed evaluating both compounds comparing drug sensitivity. Methods: Lung: ST5185B was established from a pretreated 68-year-old male; ST5431B was established from a chemo-naïve 67-year-old male; additional samples were collected during and after progression on KRASG12C-i and are currently in development. ST5489 was established from a pretreated 64-year-old female. CRC: ST4859 was established from a 57-year-old female pretreated with a KRASG12C-i; all patients received at least one cycle of KRASG12C-i therapy. Models were WES and RNA sequenced and sotorasib and adagrasib sensitivity determined. For each study, agents were dosed PO once daily at 100 mg/kg; study endpoints included tumor volume and time from treatment initiation with %T/C values reported at study completion; a %T/C of ≤ 20% was considered sensitive. Results: In all models sequencing confirmed KRASG12C and other variants previously identified with clinical studies. In vivo studies identified ST5185B as resistant to sotorasib (%T/C=70%) and adagrasib (%T/C=86%); this donor patient progressed after six weeks on KRASG12C-i therapy. ST5431B reported sensitivity to both therapies; this donor patient had a partial response (50% decrease) for six months prior to progression. ST5489 reported sensitivity to sotorasib (%T/C=14%) and adagrasib (%T/C=8%); this donor patient interrupted dosing due to KRASG12C-i intolerance and discontinued therapy, with disease progression after five weeks. ST4859 reported resistance to both drugs; this donor patient had stable disease on KRASG12C-i therapy for approximately seven months prior to progression. Ex vivo cultures and 3D-XPDX࣪ studies from all models reported sensitivity to both agents similar to in vivo studies. Summary: We developed, evaluated, and correlated in vivo sensitivity of sotorasib and adagrasib in a panel of four XPDX models established from patients receiving KRASG12C-i therapy. Studies are underway to generate drug-resistant clones of sensitive models and to elucidate mechanisms of drug resistance in ST4859. Citation Format: Johnnie Flores, Alyssa Simonson, Peter Forofontov, Anna Stackpole, Drew Rasco, Amita Patnaik, Kyriakos Papadopoulos, Teresa DesRochers, Natalie Williams, Ronald Drengler, Lon Smith, Michael J. Wick. Correlation of drug sensitivity to clinical response in XPDX models established from patients treated with KRAS-G12C-inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 412.

Adenoid cystic carcinoma (ACC) is a rare cancer that preferentially occurs in the head and neck, breast, as well as in other sites. It is an aggressive cancer with high rates of recurrence and distant metastasis. Patients with advanced disease are generally incurable due to the lack of effective systemic therapies. Activation of the master transcriptional regulator MYB is the genomic hallmark of ACC. MYB activation occurs through chromosomal translocation, copy number gain or enhancer hijacking, and is the key driving event in the pathogenesis of ACC. However, the functional consequences of alternative mechanisms of MYB activation are still uncertain. Here, we show that overexpression of MYB or MYB-NFIB fusions leads to transformation of human glandular epithelial cells in vitro and results in analogous cellular and molecular consequences. MYB and MYB-NFIB expression led to increased cell proliferation and upregulation of genes involved in cell cycle control, DNA replication, and DNA repair. Notably, we identified the DNA-damage sensor kinase ATR, as a MYB downstream therapeutic target that is overexpressed in primary ACCs and ACC patient-derived xenografts (PDXs). Treatment with the clinical ATR kinase inhibitor VX-970 induced apoptosis in MYB-positive ACC cells and growth inhibition in ACC PDXs. To our knowledge, ATR is the first example of an actionable target downstream of MYB that could be further exploited for therapeutic opportunities in ACC patients. Our findings may also have implications for other types of neoplasms with activation of the MYB oncogene. Adenoid Cystic Carcinoma Research Foundation; Swedish Cancer Society; BioCARE – a National Strategic Cancer Research Program at the University of Gothenburg; AG-Fond

We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway.

Background: Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC) consisting of an anti-HER2 (human epidermal growth factor receptor 2) antibody linked to a topoisomerase I inhibitor payload using a cleavable tetrapeptide-based linker and was recently approved for unresectable or T-DM1-refractory HER2+ breast cancer. While some mechanisms for clinical T-DM1 resistance have been identified, less is known about acquired or innate resistance to DS-8201a. We established two XPDX models of ER+/HER2+ breast cancer from tissue and fluid samples collected simultaneously from the same patient. These models designated ST4480B and ST4480C were developed and characterized for receptor expression, genomic alterations, and in vivo drug sensitivities toward multiple chemotherapies and targeted agents including DS-8201a and T-DM1. Methods: ST4480B and ST4480C were established from a 70-year-old Caucasian female with ER+/HER2+ metastatic breast cancer pretreated with chemotherapy and targeted agents including T-DM1 for nine months followed by capecitabine/trastuzumab/tucatinib combination for one year prior to sample collections. ST4480B was established from a lymph node biopsy and ST4480C from a fluid sample collected the same day; both were grown subcutaneously in female athymic nude mice supplemented with estradiol. The resulting models were passaged and receptor expression confirmed immunohistochemically; genomic analysis, including WES and RNAseq, was performed to further characterize models. For in vivo studies, both models were evaluated with several chemotherapy and targeted agents alone and in combination including: trastuzumab, pertuzumab, T-DM1, DS-8201a, neratinib, tucatinib, alpelisib, everolimus, and irinotecan. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C=— Citation Format: Johnnie R Flores, Anna Stackpole, Abimael Garza, Alexandra Ulmer, Alyssa Simonson, Kyriakos Papadopoulos, April Cabang, Jun Ma, Amita Patnaik, Drew Rasco, Amy Lang, Gladys Rodriguez, Murali Beeram, Michael J Wick. Establishment and characterization of two simultaneously developed T-DM1-resistant, ER+/HER2+ XPDX models from the same patient with differential in vivo sensitivity to trastuzumab deruxtecan (DS-8201a) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-09.

Background: Fulvestrant is a selective estrogen receptor modulator (SERM) approved as a single agent for estrogen receptor positive, HER2 negative breast cancer patients at various stages of disease and in combination with CDK4/6 inhibitors following endocrine therapy failure. Although this agent requires intramuscular injection, it has demonstrated superior activity and fewer side effects versus some oral endocrine treatments however, resistance to fulvestrant often develops. To better understand fulvestrant resistance and its utility in patients who have failed other endocrine therapies, we evaluated the agent in a panel of ER+ breast models established from patients at various stages of disease representing endocrine-sensitive and -resistant disease. Methods: Sixty-five previously developed ER+ breast XPDX models were evaluated in this study. Models were grown subcutaneously in female athymic nude mice supplemented with estradiol in drinking water when necessary. All models were characterized at early and late passages for estrogen receptor expression by immunohistochemistry and profiled using WES and RNAseq. For in vivo studies, fulvestrant was administered by subcutaneous injection at 2.5 or 5 mg per dose once weekly until study completion. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C Citation Format: April Cabang, Crystal Moreno, Johnnie R Flores, Jenna Boedeker, Alyssa Simonson, Jun Ma, Amy Lang, Gladys Rodriguez, Arthur Rosenthal, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Lon Smith, Murali Beeram, Ronald Drengler, Luis Rodriguez, Steven Abbate, Scott Ulmer, Michael J Wick. Nonclinical activity of fulvestrant in a panel of ER+ breast XPDX models representing clinically acquired and innate resistance to endocrine therapies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-11.

Background: Several CDK 4/6 inhibitors have recently been approved in combination with letrozole or fulvestrant in hormone receptor-positive breast cancer. Although this combination therapy has been found effective in some patients, resistance often develops. To aid in developing new therapies for CDK4/6 inhibitor-resistant breast cancer and better understand potential resistance mechanisms, we established a panel of nine XPDX models from eight female patients with luminal A breast cancer at time of progression following acquired resistance to CDK4/6 inhibitor therapy. These models, designated ST940C, ST2056, ST3164B, ST3164B/PBR, ST3932, ST4316B, ST4378, STF160, and STM001B were developed in athymic nude mice and characterized for receptor expression, genomic alterations, and in vivo drug sensitivity. Methods: STF160 was established from a primary biopsy and ST3932 from a metastatic soft tissue lesion; the remaining models were established from malignant fluid samples collected at various stages of treatment post CDK4/6 inhibitor response and progression. The resulting models were passaged and challenged with CDK4/6 inhibitors to confirm resistance and fulvestrant to assess sensitivity. Receptor expression was determined immunohistochemically. Genomic analysis, including WES and RNAseq, were performed to characterize models and identify mechanisms of resistance. For in vivo studies, palbociclib and abemaciclib were dosed by oral administration once daily at 50 mg/kg and fulvestrant by subcutaneous administration once weekly at 2.5 mg. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20 versus control was considered sensitive. Tumor regression (%T/C20) to palbociclib or abemaciclib and single agent fulvestrant. ST940C and ST2056 were sensitive (%T/C≤20) to tested CDK4/6 inhibitors but resistant to fulvestrant. Conclusion: We have established and characterized a panel of nine XPDX models from eight female patients with luminal A breast cancer at time of progression following acquired resistance to CDK4/6 inhibitor therapy, seven of which were found resistant to single agent palbociclib and abemaciclib and all nine to fulvestrant. This panel can be utilized as a valuable tool in better understanding CDK4/6 inhibitor resistance and in developing novel therapies for CDK4/6 inhibitor-resistant patients. Citation Format: Tatiana Hernandez, Dustin Kneifel, Alyssa Simonson, Johnnie R Flores, Sarah Quick, April Cabang, Alexandra Ulmer, Kyriakos Papadopoulos, Amy Lang, Gladys Rodriguez, Murali Beeram, Drew Rasco, Amita Patnaik, Scott Ulmer, Michael J Wick. Establishment and characterization of luminal A breast XPDX models from patients with acquired resistance to CDK 4/6 inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-06.

Pluripotent cells have been used to probe developmental pathways that are involved in genetic diseases and oncogenic events. To find new therapies that would target MYB-driven tumors, we developed a pluripotent zebrafish blastomere culture system. We performed a chemical genetic screen and identified retinoic acid agonists as suppressors of c-myb expression. Retinoic acid treatment also decreased c-myb gene expression in human leukemia cells. Translocations that drive overexpression of the oncogenic transcription factor MYB are molecular hallmarks of adenoid cystic carcinoma (ACC), a malignant salivary gland tumor with no effective therapy. Retinoic acid agonists inhibited tumor growth in vivo in ACC patient–derived xenograft models and decreased MYB binding at translocated enhancers, thereby potentially diminishing the MYB positive feedback loop driving ACC. Our findings establish the zebrafish pluripotent cell culture system as a method to identify modulators of tumor formation, particularly establishing retinoic acid as a potential new effective therapy for ACC.

Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs. Significance: Nearly 30% of endocrine therapy–resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176–93. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1047

Aberrant activation of signaling through the RAS–RAF–MEK–ERK (MAPK) pathway is implicated in numerous cancers, making it an attractive therapeutic target. Although BRAF and MEK-targeted combination therapy has demonstrated significant benefit beyond single-agent options, the majority of patients develop resistance and disease progression after approximately 12 months. Reactivation of ERK signaling is a common driver of resistance in this setting. Here we report the discovery of BVD-523 (ulixertinib), a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. In vitro BVD-523 treatment resulted in reduced proliferation and enhanced caspase activity in sensitive cells. Interestingly, BVD-523 inhibited phosphorylation of target substrates despite increased phosphorylation of ERK1/2. In in vivo xenograft studies, BVD-523 showed dose-dependent growth inhibition and tumor regression. BVD-523 yielded synergistic antiproliferative effects in a BRAFV600E-mutant melanoma cell line xenograft model when used in combination with BRAF inhibition. Antitumor activity was also demonstrated in in vitro and in vivo models of acquired resistance to single-agent and combination BRAF/MEK–targeted therapy. On the basis of these promising results, these studies demonstrate BVD-523 holds promise as a treatment for ERK-dependent cancers, including those whose tumors have acquired resistance to other treatments targeting upstream nodes of the MAPK pathway. Assessment of BVD-523 in clinical trials is underway (NCT01781429, NCT02296242, and NCT02608229). Mol Cancer Ther; 16(11); 2351–63. ©2017 AACR.

Supplemental Digital Content is available in the text., The PI3K/AKT pathway plays an important role in the initiation and progression of cancer, and the drug development efforts targeting this pathway with therapeutic interventions have been advanced by academic and industrial groups. However, the clinical outcome is moderate. Combination of inhibition of PI3K/AKT and other targeted agents became a feasible approach. In this study we assessed the combined effect of ARQ 092, a pan-AKT inhibitor, and ARQ 087, a pan-FGFR inhibitor, in vitro and in vivo. In a panel of 45 cancer cell lines, on 24% (11 out of 45) the compounds showed synergistic effect, on 62% (28 out of 45) additive, and on 13% (6 out of 45) antagonistic. The highest percentage of synergism was found on endometrial and ovarian cancer cell lines. Mutational analysis revealed that PIK3CA/PIK3R1 mutations and aberrant activation of FGFR2 predicted synergism, whereas Ras mutations showed a reverse correlation. Pathway analysis revealed that a combination of ARQ 092 and ARQ 087 enhanced the inhibition of both the AKT and FGFR pathways in cell lines in which synergistic effects were found (AN3CA and IGROV-1). Cell cycle arrest and apoptotic response occurred only in AN3CA cell, and was not seen in IGROV-1 cells. Furthermore, enhanced antitumor activity was observed in mouse models with endometrial cancer cell line and patient-derived tumors when ARQ 092 and ARQ 087 were combined. These results from in-vitro and in-vivo studies provide a strong rationale in treating endometrial and other cancers with the activated PI3K/AKT and FGFR pathways.

Introduction: Chordoma is an ultra-rare cancer found in the base of the skull and the mobile spine that originates from embryonic remnants of the notochord. It is often resistant to standard chemotherapy and most systemic anti-cancer treatment, and surgical removal followed by radiation therapy remains the mainstay of current treatment. However, due to the difficult location of chordoma, complete surgical removal is not always possible. In addition, chordoma has a high rate of metastasis and recurrence. Therefore, novel and effective treatment options for chordoma are needed. Selective inhibitor of nuclear export (SINE) compounds selinexor and eltanexor are a class of novel oral drugs that target XPO1 (exportin-1/ CRM1) and exhibit anti-cancer activity across a wide range of solid and hematological malignancies. In July 2019, selinexor was approved by the US FDA to treat patients with multiple myeloma. To investigate the preclinical efficacy and tolerability of SINE compounds in chordoma, two PDX (patient derived xenograft) mouse models of chordoma were tested. Methods: Two SINE drugs, selinexor at 5 mg/kg x 4 times a week and eltanexor at 10 mg/kg x 5 times a week, alone or in combination with bortezomib were used to treat chordoma CF466 and SF8894 PDX mouse models. CF466 PDX was derived from a patient with metastatic sacral chordoma whereas SF8894 PDX was derived from a patient with recurrent clival chordoma. Tumor volume and mouse body weight were monitored during the study. Tumors were collected at the end of 6 weeks of the study for histological, and immunohistochemistry (IHC)-based biomarker analyses. Results: Both selinexor and eltanexor demonstrated potent anti-cancer activity compared to controls. Tumor growth inhibition (TGI) of selinexor was 58% and 78% in the CF466 and SF8894 models, respectively, and TGI of eltanexor was 55% in the SF8894 model. No significant difference in body weight was observed among different treatment groups. Bortezomib did not exhibit anti-tumor activity in these models nor did it have additive/synergistic effects when combined with selinexor or eltanexor. CF466 tumors from mice treated with selinexor showed increased apoptosis, decreased cell proliferation and lowered cell density when compared with the controls. In addition, IHC analysis showed that selinexor treatment increased nuclear retention of eIF4E and tumor suppressor proteins APC, SMAD4 and FOXO3A, and decreased the expression of proteins that may play a role in chordoma tumor biology, such as SHH, GLI1 and SOX9. Conclusions: SINE compounds effectively inhibit tumor growth in PDX mouse models of chordoma. The anti-cancer effects are likely achieved through regulation of multiple signaling pathways. Further investigation of SINE compounds as treatment options for chordoma is warranted. Citation Format: Hua Chang, Leah Henegar, Trinayan Kashyap, Thaddeus J. Unger, Sharon Shacham, Josh Sommer, Patty Cogswell, Stacy Fechner, Michael J. Wick, Joan Levy, Yosef Landesman. SINE compounds demonstrated potent anti-cancer activity in PDX mouse models of chordoma [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 3086.

Mutations in ERBB2, the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not ERBB2 amplified) tumors but are rare in "HER2-positive" (ERBB2 amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of ERBB2/HER2.

Background: Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for treatment of HER2 positive (HER2+), trastuzumab-resistant breast cancers. While this agent is initially effective, resistance often develops. To aid in developing new therapies for HER2+, T-DM1-resistant breast cancer and better understanding resistance mechanisms, we established PDX models designated ST340, ST1339, and ST2167 from three patients with trastuzumab-resistant HER2+ breast cancer and sensitive to T-DM1 treatment in vivo. A cohort of each model was conditioned in vivo with chronic T-DM1 treatment to generate drug resistance. These models, designated ST340/TDR, ST1339/TDR, and ST2167/TDR were subjected to characterization and efficacy studies, and the results compared with parent models. Methods: ST340, ST1339, and ST2167 were established from patients who responded then progressed on trastuzumab and other therapies. The models were passaged, and cohorts were challenged with chronic T-DM1 treatment to produce drug resistance. The parent and resistant models were subjected to various comparative analyses including receptor expression, RNAseq, and response to various relevant therapies. Results: T-DM1-resistant models retained high HER2 expression and demonstrated receptor staining and histopathology comparable to respective parent lines. Comparison of parent and resistant clones using RNAseq identified several alterations in variants and expression. In vivo T-DM1 reported significant activity towards ST340: %T/C=3%, ST1339: %T/C=-40% and ST2167: %T/C=13% versus control, including tumor regressions in ST1339. T-DM1-resistant models reported the following values following treatment: ST340/TDR: %T/C=65%, ST1339/TDR: %T/C=58% and ST2167/TDR: T/C=91%. Conclusion: We have established and characterized three paired T-DM1-sensitive and resistant breast PDX models, which can be utilized as valuable tools in developing new therapies for T-DM1-resistant patients. Citation Format: Alyssa Moriarty, Lizette Gamez, Tomoyuki Mashimo, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Amy Lang, Ronald Drengler, Michael J Wick. Development and characterization of HER2+ T-DM1-resistant breast PDX models in athymic nude mice [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A017. doi:10.1158/1535-7163.TARG-19-A017

Background: FDA-approved CDK 4/6 inhibitors (CDK4/6i) including palbociclib, abemaciclib, and ribociclib have demonstrated clinical benefit in treating hormone receptor-positive (HR+) breast cancer. However, whether there is differential sensitivity to each therapy in vivo is unknown. In addition, whether these inhibitors demonstrate activity towards other tumor types is unclear. To better understand potential differences in CDK4/6i activity, we compared activity of each in a panel of 100 HR+/- breast cancer PDX models. To identify additional tumor types sensitive to CDK4/6i, we screened single agent palbociclib in an additional panel of 350 PDX models representing solid and hematologic malignancies. FFPE samples were collected from control and treatment groups in all studies at endpoint, and %T/C values in each model were calculated and in breast studies compared. Methods: START PDX models were established and validated as previously described and further characterized using IHC, WES, RNAseq, and drug sensitivity studies. For each CDK4/6i study, models were implanted SC in immune-deficient mice and studies initiated at 200-300 mm3 (n=1-3/grp). CDK4/6i agents were administered orally once daily at 50-75 mg/kg for a minimum of twenty-eight days or until study completion. Models tested with palbociclib included breast, lung, ovary, pancreas, head/neck, renal, gastric, uterine, colorectal, melanoma, and various hematologic malignancies. Study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20% versus control was considered sensitive. Partial tumor regression (PR) (%PR=1-99%) and complete tumor regression (CR) (%CR=100%) values versus Day 0 tumor volume of treated groups were also reported. Results: HR+/- breast models demonstrated variable sensitivity to the three CDK4/6i with HR+ models established from chemo-naïve patients reporting greatest tumor growth inhibition (TGI). Two of four HR+ ESR1 mutant models reported modest sensitivity to CDK4/6i (ST941Y537S and ST2535D538G), while ST2177Y537S was sensitive and ST2056Y537S refractory to the three therapies. Evaluation of palbociclib in additional indications reported notable TGI versus control in several indications with the following percentage of tested models reporting sensitivity: Lung: 10%, Ovary: 15%, Pancreas: 30%, Head/Neck: 35%, Renal: 45%, Gastric: 60%, Uterine: 20%, Colorectal: 45%, Melanoma: 40% and Hematologic: 15%. In addition, several indications reported tumor regressions including one CR (lung: ST1748) and partial responses in ovary, pancreas, head/neck, renal, colorectal, and melanoma. Conclusion: We evaluated and compared three FDA-approved CDK4/6i therapies in a panel of HR+/- breast PDX models and identified models with differential responses. In addition, we screened single agent palbociclib in a panel of 350 PDX models representing solid and hematologic malignancies and identified several sensitive models in multiple indications, including some with partial or complete tumor regressions. Citation Format: Lizette Gamez, Tomoyuki Mashimo, Alyssa Moriarty, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Amy Lang, Ronald Drengler, Lon Smith, Michael J Wick. PDX-based screen to evaluate and compare approved CDK4/6 inhibitors in breast cancer and determine palbociclib efficacy in additional tumor types [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A008. doi:10.1158/1535-7163.TARG-19-A008

Background: Overall, 10-30% of patients with metastatic breast cancer (BC) will develop brain metastases. Depending on the subtype of the primary breast cancer the incidence of brain metastases varies ranging from 14% (hormone receptor positive BC) to 46% (triple-negative BC) and approximately about 34% for patients with HER2-positive BC. The major impact of the blood-brain-barrier (BBB), BBB efflux pumps and the local microenvironment in the brain represent a challenge for treatment of BC brain metastasis and treatment options remain limited. The aim of this study was to develop a model system of breast cancer brain metastases for evaluation of drugs directed at BC brain metastases. Methods: Models of BC brain metastasis were established by intracranial stereotactic injection of enzymatically digested PDX tumors (ST340, ST941, ST1339, ST1616B, ST1360B, and ST3338) or by intracardiac or intracarotid injection of cell suspensions in nude mice. Contrast-enhanced T1- and T2-weighted magnetic resonance imaging (MRI) or bioluminescence imaging was used to determine tumor take and growth. Drug sensitivity to single agent trastuzumab emtansine (T-DM1) administered intravenously were performed at confirmed tumor take in a subset of the models. Mice were treated with either saline or T-DM1, 10 mg/kg/week x4 or 2 mg/kg/week x4. PET imaging with 18F-FES was applied in estrogen receptor (ER) positive models as a PD biomarker of anti-estrogen receptor endocrine therapy. Results: T-DM1 treatment in the intracranial ST1339 or ST3338 HER2 positive (3+) PDX models inhibited tumor growth and prolonged survival compared to non-treated animals. However, T-DM1 treatment only caused a modest growth delay in the ST3338 model established intracranially compared to a complete response in the model established subcutaneously. Delivery of T-DM1 across the BBB to the intracranial tumors was visualized by 64Cu-trastuzumab PET/CT and high tumor uptake was associated with a response to T-DM1 therapy in the ST1339 model. Models of BC brain metastasis were successfully established by the intracardiac or intracarotid methods for the triple negative BC cell line MDA-MB-231, the HER2 positive cell line BT474, and an ER positive cell line (ST941C). In vivo imaging with 18F-FES PET/CT was able to visualize intracranial ER positive BC tumors, and animals treated with fulvestrant exhibited reduced 18F-FES tumor uptake. Conclusion: We have established model systems of BC brain metastasis and drug efficacy evaluation. Treatment response to T-DM1 was observed in intracranial HER2 positive models and delivery of T-DM1 to the tumors was visualized by 64Cu-trastuzumab PET/CT imaging. Together, the established breast cancer brain metastases models in combination with advanced non-invasive imaging can be used as a relevant translational platform for evaluation of new drugs. Citation Format: Carsten H. Nielsen, Michael J. Wick, Lotte K. Kristensen, Henriette S. Joergensen, Maria Z. Alfsen, Mark U. Juul, Alyssa Moriarty, Kyriakos P. Papadopoulos, Andreas Kjaer. Preclinical models of breast cancer brain metastasis for drug efficacy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A011. doi:10.1158/1535-7163.TARG-19-A011

Adenoid cystic carcinoma (ACC) is a salivary gland malignancy that has no effective therapy and is caused by translocations involving MYB. A zebrafish chemical genetic screen identifies the retinoic acid class of compounds as potential MYB-inhibitory agents. Preclinical ACC mouse xenotransplantation models confirm the in vivo efficacy of retinoic acid, which represents a potential therapy for ACC., Pluripotent cells have been used to probe developmental pathways that are involved in genetic diseases and oncogenic events. To find new therapies that would target MYB-driven tumors, we developed a pluripotent zebrafish blastomere culture system. We performed a chemical genetic screen and identified retinoic acid agonists as suppressors of c-myb expression. Retinoic acid treatment also decreased c-myb gene expression in human leukemia cells. Translocations that drive overexpression of the oncogenic transcription factor MYB are molecular hallmarks of adenoid cystic carcinoma (ACC), a malignant salivary gland tumor with no effective therapy. Retinoic acid agonists inhibited tumor growth in vivo in ACC patient–derived xenograft models and decreased MYB binding at translocated enhancers, thereby potentially diminishing the MYB positive feedback loop driving ACC. Our findings establish the zebrafish pluripotent cell culture system as a method to identify modulators of tumor formation, particularly establishing retinoic acid as a potential new effective therapy for ACC.

Purpose: Antibody–drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance. Experimental Design: The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro. The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3. SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR. Results: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3, suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs. Conclusions: Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.