Mutant Patient-derived Xenograft Research Articles

PKMYT1 is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer.

Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

Abstract 1972: Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation

Abstract Genomic alterations and dysregulation of the MAPK pathway have been described in many different types of cancers. BRAF V600 point mutations and BRAF fusions, which are found in both pediatric and adult cancers, are oncogenic drivers that drive constitutive activation of the RAF pathway (Jones 2009, Yaeger 2019). Neurofibromatosis 1 (NF1) loss-of-function (LOF) mutations, which occur in many cancer types, result in decreased neurofibromin GAP function, thus activating RAS (Basu 1992). Tovorafenib is an investigational, selective, CNS-penetrant, small molecule type II RAF inhibitor which inhibits both RAF monomers and dimers and has been shown preclinically to impact the KIAA1549::BRAF fusion (Sun 2017). In this study, the impact of tovorafenib alone or in combination with MEK inhibitor, pimasertib, was explored in adult or pediatric tumor models harboring BRAF fusions or NF1-LOF mutations. At clinically relevant doses, tovorafenib treatment resulted in tumor regression in an AGK::BRAF fusion melanoma patient-derived xenograft (PDX) model in vivo. In contrast, little anti-tumor activity was observed in response to tovorafenib in vivo in two tumor models harboring NF1-LOF mutations. Ongoing PK-PD studies in both the AGK::BRAF fusion model and the NF1-LOF models will assess the extent and duration of target inhibition. In vitro, anti-proliferative activity of tovorafenib was also evaluated in NF1-LOF mutant tumor cell lines or PDX models of different tumor types, including melanoma, lung and malignant peripheral nerve sheath tumor (MPNST). While tovorafenib was potent in the BRAF V600E tumor cell line, little activity was observed in the NF1-LOF tumor cell lines. Modulation of phosphorylated-ERK (pERK) was also assessed. At very low concentrations, modest induction of pERK was observed in the NF1-LOF tumor cell lines, with inhibition at higher concentrations. In vitro combination studies were conducted using pimasertib and tovorafenib, or a tool compound. Synergy was observed in a NF1-LOF embryonal rhabdomyosarcoma PDX model ex vivo and a NF1-LOF MPNST cell line in vitro, suggesting that vertical pathway inhibition is impactful in the NF1-LOF mutant setting. Tovorafenib is currently being evaluated as a monotherapy in relapsed or progressive pediatric low-grade glioma (pLGG) harboring RAF alterations (NCT04775485) and in combination with pimasertib in patients ≥12 years of age with recurrent, progressive, or refractory solid tumors harboring MAPK pathway alterations (NCT04985604). Ongoing preclinical translational work will continue to explore potential biomarker-defined tumor indications for these agents. Citation Format: Shubhra Rastogi, Sam Perino, Madhu Lal-Nag, Yujin Wang, Samuel C. Blackman, Eleni Venetsanakos. Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation [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 1972.

ASCL1 Drives Tolerance to Osimertinib in EGFR Mutant Lung Cancer in Permissive Cellular Contexts.

Analysis of residual disease following tyrosine kinase inhibitor treatment identified heterogeneous and context-specific mechanisms of drug tolerance in lung cancer that could lead to the development of strategies to forestall drug resistance. See related commentary by Rumde and Burns, p. 1188.

Abstract B124: MYTX-011 is a highly internalized ADC with anti-tumor activity across a spectrum of NSCLC preclinical models with various levels of cMET expression

Abstract cMET overexpression occurs in non-small cell lung cancer (NSCLC) and antibody-drug conjugates targeting cMET (cMET-ADCs) have been developed to treat cMET+ NSCLC tumors irrespective of dependency on cMET signaling. cMET-ADCs have shown promising clinical activity, but largely in a subset of NSCLC patients whose tumors express the highest cMET levels. MYTX-011, an investigational, pH-sensitive ADC incorporating the clinically validated vcMMAE linker-payload, was specifically designed to potentially benefit a broader population of patients including those whose tumors express lower cMET levels. We’ve previously demonstrated that pH-sensitive cMET binding of MYTX-011 drives higher internalization in cMET+ tumor cells, and greater cytotoxicity against a panel of cMET+ cancer cell lines in vitro. MYTX-011 also shows superior efficacy in NSCLC xenograft models with high or moderate cMET expression levels when compared to the matched non-pH-sensitive Parent ADC or a Benchmark ADC generated by hinge conjugation of a clinical stage anti-cMET ADC antibody to vcMMAE. Here we demonstrate that the MYTX-011 monoclonal antibody, which is engineered to selectively and rapidly lose affinity at the acidic pH of endolysosomes, rapidly internalizes and accumulates in cells expressing moderate cMET levels compared to the matched parent antibody. MYTX-011 in vitro cytotoxicity correlated with cMET expression levels and conferred sensitivity to cells that were otherwise insensitive to ADCs generated with the parent or Benchmark antibodies. Importantly, cells that were only sensitive to MYTX-011, but not matched controls, expressed low and moderate levels of cMET. Furthermore, MYTX-011 was active against the low cMET-expressing NCI-H2122 xenograft model in vivo whereas the Benchmark ADC was inactive. In the moderate cMET-expressing NCI-H1975 xenograft model, MYTX-011 was more active than both the Benchmark ADC and a second benchmark ADC generated by hinge conjugation of a clinical stage biparatopic anti-cMET ADC antibody to a maytansinoid linker/toxin. MYTX-011 activity was further evaluated against a panel of NSCLC patient derived xenograft (PDX) models with relevant histology, cMET expression, and genetic profiles. MYTX-011 was highly active in cMET+ squamous and adenocarcinoma PDX, EGFR WT and mutant PDX, in a PDX model with MET ex14 skipping mutation, and in PDX with heterogenous cMET expression. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET+ NSCLC malignancies than other cMET-ADCs. Citation Format: William C Comb, Deepak Kanojia, Federico Colombo, Vijay Ramesh, Nimish Gera, Thomas Chittenden, Brian Fiske. MYTX-011 is a highly internalized ADC with anti-tumor activity across a spectrum of NSCLC preclinical models with various levels of cMET expression [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B124.

Giredestrant reverses progesterone hypersensitivity driven by estrogen receptor mutations in breast cancer.

<i>ESR1</i> (estrogen receptor 1) hotspot mutations are major contributors to therapeutic resistance in estrogen receptor-positive (ER⁺) breast cancer. Such mutations confer estrogen independence to ERα, providing a selective advantage in the presence of estrogen-depleting aromatase inhibitors. In addition, <i>ESR1</i> mutations reduce the potency of tamoxifen and fulvestrant, therapies that bind ERα directly. These limitations, together with additional liabilities, inspired the development of the next generation of ERα-targeted therapeutics, of which giredestrant is a high-potential candidate. Here, we generated <i>Esr1</i> mutant-expressing mammary gland models and leveraged patient-derived xenografts (PDXs) to investigate the biological properties of the <i>ESR1</i> mutations and their sensitivity to giredestrant in vivo. In the mouse mammary gland, <i>Esr1</i> mutations promote hypersensitivity to progesterone, triggering pregnancy-like tissue remodeling and profoundly elevated proliferation. These effects were driven by an altered progesterone transcriptional response and underpinned by gained sites of ERα-PR (progesterone receptor) cobinding at the promoter regions of pro-proliferation genes. PDX experiments showed that the mutant ERα-PR proliferative program is also relevant in human cancer cells. Giredestrant suppressed the mutant ERα-PR proliferation in the mammary gland more so than the standard-of-care agents, tamoxifen and fulvestrant. Giredestrant was also efficacious against the progesterone-stimulated growth of <i>ESR1</i> mutant PDX models. In addition, giredestrant demonstrated activity against a molecularly characterized <i>ESR1</i> mutant tumor from a patient enrolled in a phase 1 clinical trial. Together, these data suggest that mutant ERα can collaborate with PR to drive protumorigenic proliferation but remain sensitive to inhibition by giredestrant.

Abstract 333: Activity of the erythropoietin-producing hepatocellular A2 receptor (EphA2) targeting Bicycle® Toxin Conjugate (BTC™) BCY6033 in EGFR inhibitor resistant non-small lung cancer (NSCLC) patient derived xenografts

Abstract Introduction: EphA2 regulates cell migration, adhesion, proliferation and differentiation, and is overexpressed in human cancers which have been shown to correlate with tumor progression. BCY6033 contains a bicyclic peptide targeting EphA2, linked to the cytotoxin Monomethyl auristatin E via a molecular spacer and cleavable linker designed to target EphA2 expressing tumors. BCY6033 is analogous to BT5528, a BTC™ currently in phase I/II clinical trial in patients with advanced malignancies associated with EphA2 expression. Recent literature indicate that tumor EphA2 expression may be up regulated in EGFR mutant lung models following 1st, 2nd and possibly 3rd generation EGFR inhibitors. Here we report on EphA2 expression in well characterized and clinically annotated patient derived xenografts (PDX) of NSCLC including EGFR mutant NSCLC derived either from erlotinib or osimertinib resistant patients. In vivo activity of BCY6033 is presented in two EphA2 expressing PDX models. Experimental procedures: In vivo, an initial tolerability study was conducted with once weekly administration of BCY6033 by intravenous injection in NSG mice. The activity of BCY6033 was evaluated in EphA2 expressing EGFR mutant PDX models, DFCI-161 and DFCI-220 in NSG mice. A TMA was constructed containing FFPE derived from 69 PDX models (NSCLC n=61, SCLC-transformed n=5, de novo SCLC n=3). Of the 69 models, 35 were EGFR mutant. The TMA was subsequently stained via immunohistochemistry (IHC) using an α-EphA2 (R&amp;D Systems) primary antibody. Tumor membranous H-score was assigned by a pathologist and a score of ≥50 was considered positive. Results: Of the 69 PDXs, 16 were determined to be EphA2 positive. No correlation with genotype or clinical history was observed with EphA2 expression. EphA2 IHC was repeated on at least 5 different tumors for 5 PDX models with fast and robust in vivo growth latency. Two models, DFCI-161 and DFCI-220 with the most reproducible EphA2 staining were selected. Treatment with BCY6033 showed that a dose of 3 mg/kg once weekly administered intravenously was well tolerated in female NSG mice. BCY6033 treatment led to significant tumor growth inhibition with &amp;gt;80% tumor regressions in both DFCI-161 and DFCI-220 PDX models. Re-challenge of tumor out-growth showed that they retain sensitivity to BCY6033 treatment. Conclusion: BCY6033 is a potentially promising drug effective against EphA2 expressing PDX models. Future studies and clinical trials will seek to determine the efficacy of BT5528 in EphA2 expressing tumors. Citation Format: Kenneth Ngo, Elena V. Ivanova, Tyler J. Teceno, Carly Campbell, Johanna Lahdenranta, Stephen J. Blakemore, Gavin Bennett, Pasi A. Jänne, Cloud P. Paweletz, Prafulla C. Gokhale. Activity of the erythropoietin-producing hepatocellular A2 receptor (EphA2) targeting Bicycle® Toxin Conjugate (BTC™) BCY6033 in EGFR inhibitor resistant non-small lung cancer (NSCLC) patient derived xenografts [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 333.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

SUMMARYRAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Analyses of DNA repair protein expression in BRCA1 mutant patient-derived xenografts

<h3>Objectives:</h3> To evaluate the potential of DNA damage response protein expression and foci formation to serve as biomarkers of resistance to platinum chemotherpay or Poly-(ADP)-Ribose Polymerase Inhibitors (PARPi) using immunohistochemistry (IHC). <h3>Methods:</h3> A collection of four patient-derived xenografts (PDX) mouse models with <i>BRCA1</i> mutations in exon 11, a <i>BRCA1</i> exon 13 mutant, and a <i>BRCA1</i> wild-type tumor were analyzed. <i>BRCA1</i> exon 11 mutants included: PDX196, mutation: c1961delA; PDX124, mutation: c196delA; PDX017, mutation: c1105_1106insTC; PDX056, mutation: c895_896delGT; <i>BRCA1</i> WT control: PDX036 and <i>BRCA1</i> exon 13 mutant: PDX1126, mutation: c4327C>T. Mice harboring PDX tumors were subjected to vehicle, PARPi (rucaparib), or platinum (cisplatin) treatment. Passaging and re-treatment of tumors was performed until minimal therapy response. Formalin-fixed paraffin embedded tumor samples was prepared for IHC analyses. IHC protocols were optimized to evaluate expression and foci formation for the following proteins: phosphorylated-RPA32, Rad51, phosphorylated-53BP1, 53BP1, <i>BRCA1,</i> Ki67. <h3>Results:</h3> <i>BRCA1</i> foci formation was observed by IHC in PARPi and cisplatin resistant tumors. Reversion mutations were not detected and <i>BRCA1</i> protein expression was due to increased levels of <i>BRCA1</i>-D11q. Increased RPA32 and RAD51 foci were also observed in several therapy resistant PDX models, which indicate active HR repair. Interestingly, acquired resistance to cisplatin and PARPi was associated with a reduction in 53BP1 and phosphorylated-53BP1. Finally, PARPi and cisplatin resistant tumors exhibited similar levels of <i>BRCA1</i> and RAD51 foci. <h3>Conclusions:</h3> <i>BRCA1</i>-D11q protein expression correlates with platinum and PARPi response in several <i>BRCA1</i> exon 11 mutant PDX models. We predict that increased <i>BRCA1</i>-D11q in combination with decreased 53BP1 expression drives RPA and RAD51 foci, resulting in active homologous recombination and therapy resistance. Our results suggest that IHC can be utilized for detection of DNA damage repair protein expression as well as foci formation, and could lead to the development of biomarkers that are predictive of therapy response.

Abstract 3004: Establishment of KRAS G12C mutant patient-derived xenograft (PDX) models for pre-clinical evaluation of KRAS G12C targeted anticancer therapy

Abstract KRAS is one of the most prevalent oncogenes in human cancers. The G12C mutation results in an amino acid substitution at position 12 in KRAS, from a glycine (G) to a cysteine (C), lead to accretion of GTP-bound activated state KRAS and activation of downstream signaling pathways. In non-small cell lung cancer (NSCLC), KRAS p.G12C is the most common mutation, comprising nearly half of all KRAS mutations. Recent breakthrough work led to the development of selective inhibitors targeting the KRAS G12C mutation, which have shown promise in early clinical trials. However, the therapeutic benefit of targeted therapies can be impaired by intrinsic or acquired resistance mechanisms, suggesting an urgent need of combinatorial strategies to overcome the resistance to drugs targeting KRAS G12C.Here we report on the establishment of a panel of KRAS G12C mutant PDX models, encompass non-small cell lung cancer, colorectal cancer and gastric cancer. The activity of AMG510, a selective KRAS G12C inhibitors in clinical trial, was characterized in these PDX models. Data showed that the validated KRAS G12C mutant models display a range of sensitivity to single treatment of AMG510. Given that in clinic only 50% of NSCLC patients with KRAS G12C mutation would benefit from KRAS G12C targeted inhibitors, in order to provide an insights for clinical therapeutic strategy, we investigated the combinatorial strategies in NSCLC PDX models: (1) AMG510 combined with Opdivo in KRAS G12C/CD274 amplification models in hPBMC humanized mice; (2) AMG510 combined with EGFR inhibitor in KRAS G12C/EGFR overexpression models. The possible mechanisms of combination effect were discussed. Altogether, the panel of KRAS G12C mutant PDX models are valuable for the pre-clinical evaluation of KRAS G12C targeted anti-cancer therapies. Citation Format: Wenting Shi, Xuzhen Tang, Jingying Zhang, Xinhong Kuang, Yan Zhang, Jingjing Wang, Qingyang Gu, Qunsheng Ji. Establishment of KRAS G12C mutant patient-derived xenograft (PDX) models for pre-clinical evaluation of KRAS G12C targeted anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3004.

Binimetinib activity in PIK3R1-mutant patient-derived xenografts (PDX) implanted into immunodeficient mice.

e13062 Background: The PIK3R1 gene is genetically altered in ̃3% of breast cancers in the Western and ̃17% in the Eastern worlds. We recently reported that breast cancer cells lacking protein expression of PIK3R1 had elevated levels of activated MEK, sensitizing them to the MEK inhibitor trametinib. To better understand whether MEK inhibition is a therapeutic option for breast cancer patients with mutated PIK3R1, we tested the newer generation MEK inhibitor, binimetinib, in a PDX mouse model. Methods: Two PDx models (table) were tested in athymic nude-foxn1nu (immune-compromised) mice. Both patients had had metastatic, grade 3 breast cancer and expressed moderate RNA levels of PIK3R1. Results: Body weights of treatment arms were unchanged throughout the experiment. The TNBC PDx study was halted early at 13 days because tumors reached the predefined maximum size of 1500 mm2. The binimetinib treated mice experienced slowed tumor growth (522.8 +/- 172.8%) compared to the vehicle control (720.7 +/- 229.1%), (p=0.0693). In the HR+ PDx model, the combination of binimetinib plus tamoxifen resulted in a statistically significant decrease (371.5 +/- 108.1%) in tumor volume compared to tamoxifen alone (525.0 +/- 154.1%; p = 0.0302). Conclusions: Our previous and current results suggest that mutation in PIK3R1 sensitizes cells to MEK inhibitors. Binimetinib inhibited growth in both models. This inhibition was of borderline significance in the TNBC model and was statistically significant in the ER+, HER2- model. To the best of our knowledge, these results provide the first evidence of binimetinib efficacy in PIK3R1-mutant, HR+, HER2- PDX immunodeficient mice. Plans for a phase II trial in patients with PIK3R1-mutant, HR+, HER2- breast cancer are underway.[Table: see text]

Abstract PS4-13: Irreversible inhibition of HER2 activating mutations with neratinib enhances the pre-clinical efficacy of trastuzumab emtansine and trastuzumab deruxtecan

Abstract Background: HER2 activating mutations occur in 2-5% of metastatic breast cancer (MBC) patients, and three phase II or basket clinical trials have shown that the irreversible pan-HER tyrosine kinase inhibitor, neratinib, has good single agent efficacy for HER2 mutated MBC patients. Current trials are combining neratinib with other targeted therapies to increase response rate and progression free survival for these patients. Methods: We established patient derived xenografts (PDX) and organoids from two patients with HER2 mutated, non-amplified MBC and used them to test neratinib with the antibody drug conjugates (ADC’s), trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), both in 3D culture and in vivo. Real time, in vivo uptake of these ADC’s was visualized with a near infrared fluorophore. Results: PDX lines WHIM51 and WHIM64 were established from ER+, HER2 non-amplified MBC patients that had HER2 activating mutations. WHIM51 has HER2 exon 20 insertion mutation at amino acid 776 (ERBB2 A775_G776insYVMA) and WHIM64 has a HER2 L869R missense mutation, both of which are located in the HER2 tyrosine kinase domain. Both of these HER2 mutations have been previously characterized and are known activating mutations. Organoids were established from both PDX’s and were grown in 3D culture. Drug combination testing of neratinib with T-DM1 in 3D culture showed strong synergy and the mechanism was explored. We demonstrate that neratinib and other irreversible HER2 inhibitors increase the endocytic uptake of T-DM1, but this effect does not occur with the reversible HER2 inhibitors, tucatinib and lapatinib. Real time, in vivo uptake of T-DM1 was measured by labeling the ADC with a near infrared fluorophore and we observed statistically significant increase in T-DM1 uptake with neratinib pre-treatment. Combining neratinib with T-DM1 increased apoptosis at day 3 post-treatment and enhanced tumor shrinkage. With the FDA approval of T-DXd at the end of 2019, we hypothesized that this same mechanism may apply to neratinib combined with T-DXd. We have tested both the combinations of neratinib + T-DXd and neratinib + T-DM1 in vivo in both HER2 mutant PDX’s and observed statistically significant tumor regression with the neratinib + ADC combinations as compared to either T-DXd or T-DM1 on its own. Conclusions: Neratinib increases the endocytosis of trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), thereby increasing tumor cell kill and causing greater tumor regression in HER2 mutated MBC. These data provide preclinical justification for trials of neratinib plus HER2 ADCs including T-DXd or T-DM1 in HER2 mutant or HER2+ MBC. Further, this mechanism of neratinib stimulated HER2 endocytosis may also apply to HER2 low MBC. Citation Format: Ron Bose, Shunqiang Li, Tina M. Primeau, Maureen K. Highkin, Ashley R. Tipton, Nagalaxmi Vemalapally, Xuefeng Gao, Gail Sudlow, Irmina Diala, Yu Tao, Jingqin Luo, Ian Hagemann, Chieh-Yu Lin, Richard P. Bryce, Alshad S. Lalani, Samuel Achilefu, Cynthia X. Ma. Irreversible inhibition of HER2 activating mutations with neratinib enhances the pre-clinical efficacy of trastuzumab emtansine and trastuzumab deruxtecan [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-13.

RON signalling promotes therapeutic resistance in ESR1 mutant breast cancer

BackgroundOestrogen Receptor 1 (ESR1) mutations are frequently acquired in oestrogen receptor (ER)-positive metastatic breast cancer (MBC) patients who were treated with aromatase inhibitors (AI) in the metastatic setting. Acquired ESR1 mutations are associated with poor prognosis and there is a lack of effective therapies that selectively target these cancers.MethodsWe performed a proteomic kinome analysis in ESR1 Y537S mutant cells to identify hyperactivated kinases in ESR1 mutant cells. We validated Recepteur d’Origine Nantais (RON) and PI3K hyperactivity through phospho-immunoblot analysis, organoid growth assays, and in an in vivo patient-derived xenograft (PDX) metastatic model.ResultsWe demonstrated that RON was hyperactivated in ESR1 mutant models, and in acquired palbociclib-resistant (PalbR) models. RON and insulin-like growth factor 1 receptor (IGF-1R) interacted as shown through pharmacological and genetic inhibition and were regulated by the mutant ER as demonstrated by reduced phospho-protein expression with endocrine therapies (ET). We show that ET in combination with a RON inhibitor (RONi) decreased ex vivo organoid growth of ESR1 mutant models, and as a monotherapy in PalbR models, demonstrating its therapeutic efficacy. Significantly, ET in combination with the RONi reduced metastasis of an ESR1 Y537S mutant PDX model.ConclusionsOur results demonstrate that RON/PI3K pathway inhibition may be an effective treatment strategy in ESR1 mutant and PalbR MBC patients. Clinically our data predict that ET resistance mechanisms can also contribute to CDK4/6 inhibitor resistance.

TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 &amp;gt;3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

Abstract 6216: Therapeutic potential of IAP inhibitor APG-1387 in combination with PARP- or MEK-targeted therapy, or chemotherapy in pancreatic cancer

Abstract APG-1387 is a small molecule antagonist of the inhibitor of apoptosis proteins (IAPs) which blocks the activity of IAPs family proteins (XIAP, cIAP-1, cIAP-2, and ML-IAP) and consequently induces apoptosis. It is currently in phase I clinical development in patients with solid tumors, including pancreatic cancer. Targeting IAPs has been reported to effectively inhibit tumor growth in the preclinical models of pancreatic cancer. Currently, PARP and MEK inhibitors are also in clinical development for pancreatic cancer with BRCA1/2 or KRAS mutations. To further explore the therapeutic potential of APG-1387 in combination with PARP or MEK inhibitors, a genomic biomarker guided avatar mouse trial (n=2) using pancreatic cancer patient-derived xenografts (PDX) was conducted to evaluate antitumor activity of APG-1387 in combination with (i) the PARP inhibitor, olaparib, in PDXs carrying BRCA1/2 mutations, and (ii) the MEK inhibitor, trametinib, in PDXs carrying KRAS mutations. The results revealed that APG-1387 improved the antitumor activity of olaparib in 4 out of 6 (67%) BRCA1/2 mutant PDXs with a 44% reduction of T/C (%) value (T, treatment group, C, vehicle control), including one partial tumor regression (PR). In combination with trametinib, the antitumor activity was improved in 4 out of 9 (44%) KRAS mutant PDXs, showing a 34% reduction in the T/C value. Furthermore, synergy of the combinations of APG-1387 with PARP or MEK inhibitor was confirmed in a follow-up efficacy study (n=5) using a PDX model carrying both BRCA2 and KRAS mutations. The molecular mechanisms underlying the synergy are currently being investigated. In summary, our results suggest that APG-1387 is a potential agent for pancreatic cancer treatment, ascribing to its potential synergistic antitumor effect by combining with either PARP inhibitors in BRCA1/2 mutant or MEK inhibitors in KRAS mutant pancreatic cancers. Overall, our data provide the scientific rationale for the future clinical development of these combinations in pancreatic cancer patients with distinct genomic alterations. Citation Format: Qiang Li, Douglas D. Fang, Shoulai Gu, Dajun Yang, Yifan Zhai. Therapeutic potential of IAP inhibitor APG-1387 in combination with PARP- or MEK-targeted therapy, or chemotherapy in pancreatic cancer [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 6216.

Abstract 3001: Drug resistant PDX models for new drug discovery

Abstract Drug resistant is one of the biggest challenge for cancer therapy. How to understand the mechanism and to overcome the drug resistant with new therapeutics represent the hot focus for oncology drug discovery. Drug resistant patient-derived xenograft (PDX) models derived from surgery samples, biopsy samples, hydrothorax or ascetic fluid provides unique tool for new therapeutics R&amp;D. We have successfully established an array of drug resistant PDX models. Representative drug resistant PDX includes: clinically AZD9291-resistant EGFR triple mutant (Ex19del, T790M, C797S) PDX model. In vivo efficacy results show that this triple mutant NSCLC PDX model was resistant to Erlotinib and AZD9291 and sensitive to the combination of Cetuximab and Brigatinib. We also established CD74-ROS1 fusion and G2032R mutant NSCLC PDX model; EML4-ALK fusion and L1196M mutant NSCLC PDX model; Olaparib resistant Ovarian cancer PDX model; Heceptin resistant gastric PDX model; Rituximab and ABT-199 resistant DLBCL PDX model, and so on. In conclusion, these precious PDX models provide insight into resistance mechanisms as well as serve as models to develop new therapeutics drug candidates that overcome the drug resistant. Citation Format: Feifei Zhang, Yuan Long, Danyi Wen. Drug resistant PDX models for new drug discovery [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 3001.

Abstract 4127: Niraparib is active against prostate xenograft models that are wild type or defective for DNA damage repair genes

Abstract Patients with metastatic prostate cancer may respond well to first- and second-generation androgen receptor (AR)-targeted therapies, but all will eventually experience disease progression, indicating a need for alternative therapies. A subset of these patients bears prostate tumors harboring mutations that cause DNA repair defects (DRD). Alterations in DRD genes, such as breast cancer (BRCA)2, render tumors sensitive to drugs that inhibit poly (ADP-ribose) [PAR] polymerase-1 (PARP1), thereby inducing synthetic lethality. Clinical study results of PARP inhibitors tested in late stage prostate cancer have confirmed this hypothesis. However, not all tumors with mutations in DRD genes respond to PARP inhibition, and it is possible that some patients with DRDwt prostate tumors could benefit from treatment. To better understand the basis for response to PARP inhibition, nonclinical studies evaluated the efficacy of niraparib in genetically-profiled prostate xenograft models and advanced prostate cancer patient-derived xenograft (PDX) models. The models tested included BRCA2 biallelic mutant, ataxia-telangiectasia mutated (ATM) biallelic mutant, BRCA2 monoallelic mutant and DRDwt tumor models. The two BRCA2 biallelic mutant PDX models showed variable responses to niraparib, with significant tumor growth inhibition (TGI) seen in the LuCaP 174.1 PDX, but more modest TGI observed in LuCaP 96CR. This result is similar to the clinical experience, in which many, but not all, patients with biallelic BRCA2 mutant tumors respond to PARP inhibition. BRCA2 monoallelic mutant models did not respond to PARP inhibition, suggesting that a reduction in functional BRCA2 was insufficient to result in PARP inhibitor sensitivity. In contrast, two ATM biallelic mutant models responded to niraparib treatment with significant TGI and survival benefit. Finally, DRDwt models showed distinct patterns of response to niraparib, in that several models showed significant TGI/prolonged survival, while the majority of DRDwt models did not respond. IHC studies indicate that niraparib treatment in responsive tumor models is associated with increased levels of caspase-3, histone γ-H2AX phosphorylation (indicating increased DNA double-stranded breaks), and reduced cell proliferation. Taken together, these studies support the hypothesis that niraparib can provide clinical benefit to prostate cancer patients whose tumors are DRDmutant. Further research is warranted to understand the efficacy of niraparib in DRDwt prostate tumors. Citation Format: Linda A. Snyder, Krista Menard, Georges Habineza Ndikuyeze, Holly M. Nguyen, Anna Hughes, Lorraine Angelillo, Gerald Chu, Jenny Driscoll, Rebecca Hawkins, Eva Corey. Niraparib is active against prostate xenograft models that are wild type or defective for DNA damage repair genes [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 4127.

Abstract 557: Novel targeted combination therapies active in KRAS mutant non-small cell lung cancer (NSCLC) identified using patient-derived xenografts (PDX)

Abstract PDXs recapitulate histologic features, gene expression patterns, and genomic alterations in human primary tumors, and thus have emerged as robust preclinical models for drug development, molecular characterization of cancers, identification of biomarkers, and strategic development of precision therapy. We tested multiple combinations of small molecule targeted drugs selected on the basis of known NSCLC vulnerabilities for efficacy in NSCLC PDXs with known genotypes. We determined in vivo treatment responses to single agent and combination therapies for pathway targeted therapeutic agents, including the MEK inhibitor trametinib, the MDM2 inhibitor KRT-232, the BCL2/BCL-XL inhibitor navitoclax, and their combinations in 8-23 molecularly annotated NSCLC PDX models. Mice (n=3-5/group) were enrolled into treatment individually when tumors reached 200 mm3 in size, and were treated 5 days/week for 3 weeks. Tumor growth was monitored 2-3 times/week. We used the following criteria to determine treatment responses: 1) Tumor Regression (or partial response): tumor regression ≥ -30% based on tumor volume changes calculated by AUC0-21day or at day 21 after treatment start when compared with baseline (beginning of treatment at day 0); 2) Tumor Growth Inhibition (or stable disease): Tumor growth was significantly suppressed when compared with control (P &amp;lt; 0.05), but no tumor regression was observed, or tumor regression was less than -30% based on tumor volume changes calculated by AUC0-21day or at day 21 after treatment start when compared with baseline; 3) Resistance: Tumor volume changes calculated by AUC0-21day was not significantly different from control group (P&amp;gt;0.05). Our results showed that KRT-232 alone resulted in 15.8% (3/19) tumor regression and 26.3% (5/19) tumor growth inhibition, all in TP53 wild type PDXs. Trametinib alone induced 10% (2/20) tumor regression and 50% (10/20) growth inhibition, respectively. 80% (8/10) of KRAS mutant PDXs responded to trametinib treatment with tumor regression (1/10) or growth inhibition (7/10). Combination therapies of trametinib plus KRT-232 and trametinib plus navitoclax led to improved in vivo anticancer activity over single agent activity in a subset PDX models with KRAS mutations. Tumor regression was observed in 26% (6/23) and 50% (5/10) of trametinib plus KRT-232 and trametinib plus navitoclax treatment groups, respectively. Navitoclax alone did not induce tumor regression in 8 PDX models tested, and navitoclax plus KRT-232 did not lead to significant improvement in activity over single agents in 11 PDXs. Our results show that combination therapies of trametinib plus KRT-232 or navitoclax result in improved efficacy in a subgroup of NSCLC PDX models with KRAS mutations. Clinical trials with these targeted drug combinations in NSCLC are warranted. Citation Format: Xiaoshan Zhang, Ran Zhang, Huiqin Chen, Li Wang, Chenghui Ren, Shuhong Wu, Min Jin Ha, Jeffrey Morris, Yuanxin Xi, Jing Wang, Don L. Gibbons, John V. Heymach, Funda Meric-Bernstam, John Minna, Stephen G. Swisher, Jack A. Roth, Bingliang Fang. Novel targeted combination therapies active in KRAS mutant non-small cell lung cancer (NSCLC) identified using patient-derived xenografts (PDX) [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 557.

TMOD-41. ORTHOTOPIC PATIENT-DERIVED XENOGRAFT MODELS OF BRAIN METASTASIS: PLATFORMS FOR PRECISION ONCOLOGY AND UNDERSTANDING TUMOR BIOLOGY

Abstract RATIONALE Brain metastasis (BrM) occurs in 10–20% of cancer patients and results in median survival times of less than 1 year. In order to accurately develop novel treatment strategies, there is an urgent need to establish animal models of BrM that resemble the human disease. OBJECTIVES: We sought to establish orthotopic patient-derived xenografts (PDX) models of BrM from diverse solid cancers to understand biological characteristics of BrM and to test novel drug candidates in their ability to treat BrM. METHODS 35 PDXs were established by subcutaneous or mammary fat pad implantation and by intracranial injection. PDXs were validated by immunohistochemistry of routine pathological markers in patient specimens and matched primary and intracranial PDXs. PDXs were applied for precision medicine in class II BRAF mutant tumours treated with targeted therapy and in the pre-clinical development of a novel therapeutic agent, DZ-2384, which may have applications in treating BrM. They were also used to understand the underlying biology of leptomeningeal dissemination from parenchymal BrM. RESULTS PDXs reveal strong similarity to patient specimens by IHC staining. Class II BRAF mutant PDXs are optimally sensitive to BRAF and MEK inhibitors compared to either agent alone. DZ-2384 is effective in slowing the progression of breast cancer BrM. PDXs that most efficiently invade the leptomeninges display loss of E-Cadherin expression, suggesting the role of an epithelial-mesenchymal transition in this process. CONCLUSIONS PDXs of BrM represent important models that can be employed to test novel therapeutics and to improve understanding of molecular mechanisms engaged by BrM.

P2.14-40 Tumor-Stromal Microenvironment Interactions in a PDX Model of EGFR TKI Drug Tolerance

Sixty to eighty percent of advanced stage lung adenocarcinoma patients with sensitizing epidermal growth factor receptor (EGFR) mutated tumors respond to EGFR tyrosine kinase inhibitors (TKIs). However, the vast majority of patients eventually progress due to acquired resistance. In vitro evidence suggests that minor populations of drug tolerant cells (DTCs) may be important for tumors surviving TKI. These studies cannot investigate changes in non-cancerous cell populations found within tumors. Yet, stromal cells have been implicated in protecting cancer cells from treatment-induced death and early stage lung adenocarcinomas responding to neoadjuvant EGFR TKI exhibited DTCs within large areas of fibrosis (NCT00188617). We hypothesize that molecularly characterizing DTCs in vivo in comparison to an untreated tumor in a patient-derived xenograft (PDX) model may delineate stromal changes that sustain DTCs, and potentially mimic clinical events. DTCs were harvested after one month of chronic erlotinib exposure in a lung adenocarcinoma PDX model harboring an exon 19 deletion; an untreated baseline (BL) tumor was also harvested. Histological characterization and single-cell RNA-sequencing (scRNA-seq) of DTCs and BL tumors were compared. ScRNAseq cell-types were assigned using reference component analysis. RNA expression levels of receptor/ligands were explored in cell populations. Post-erlotinib treatment, cell-type proportions within the tumor shifted dramatically, with substantially fewer cancer cells and more fibroblasts, mesenchymal stem cells (MSCs), and natural killer cells (NKCs). Two antigen presenting cell transcriptomic states (APC1 and APC2) were identified in both DTC and BL tumors: APC1s exhibited translation-related gene expression profiles while APC2s exhibited immune-response profiles. BL tumors contained mostly APC1s, whereas DTC tumors exhibited more equal proportions of both APC types. Expression profiles for some cell-types also shifted after treatment. Fibroblasts and NKCs exhibited shifts toward more inflammatory and immune-responsive expression profiles post-treatment. Fibroblasts and endothelial cells demonstrated gene expression shifts towards decreased angiogenesis and vasculature development. Paired ligand-receptor interactions between cancer-stromal cells were increased or decreased congruently post-treatment. Specifically, fibroblasts exhibited a shift from alpha-SMA+ myofibroblastic to more IL6+ inflammatory phenotypes, by mRNA and equivalent immunohistochemistry, post-treatment. Cancer cells exhibited a reciprocal increase in IL6R receptor expression post-treatment. Using an EGFR mutant PDX model sensitive to EGFR TKIs, we see substantial post-treatment changes after chronic TKI exposure in non-cancerous (stromal) cell population composition involving their proportions, expression profiles, and their inferred communication with cancer cells. Understanding these potentially protective shifts in non-cancerous cell populations post-TKI-treatment may help identify clinically-relevant mechanisms of drug tolerance.

Abstract 3870: The addition of CB-839 to temozolomide significantly reduces glioma aspartate and glutamate in an IDH mutated patient derived glioma xenograft model

Abstract Background: IDH mutated gliomas are critically dependent on glutaminase for glutamate biosynthesis. CB-839 is a novel glutaminase-1 inhibitor which has demonstrated efficacy in both genetically engineered and patient derived IDH mutated glioma cells, especially in combination with radiation. These preclinical studies evaluate the pharmacodynamic and pharmacokinetic impact of combining CB-839 with TMZ in patient derived xenograft (PDX) glioma models. Methods: GBM164 is a PDX model derived from an IDH1 mutant, 1p/19q non-codeleted glioma. D- and L-2HG levels in untreated GBM164 tumors were compared to GBM6 tumors (IDH wildtype glioma PDX) by GC/MS. Athymic nude mice bearing GBM164 flank tumors were treated with CB-839 (200 mg/kg PO BID x 9 doses) and/or temozolomide (50 mg/kg PO daily x 5 doses) and sacrificed four hours after the last dose to harvest plasma, normal brain and flank tumor. Tumor metabolomics were assessed by GC/MS. DNA damage signaling in tumors was assessed by Western blotting for KAP1 / Chk1 / Chk2 phosphorylation and H2AX / Rad51. Tumor, plasma and brain pharmacokinetics were assessed by LC/MS/MS. Results: Total 2HG levels in GBM164 were 18-fold higher than GBM6. CB-839 monotherapy reduced tumor glutamate (18% reduction, p = 0.15) and aspartate (30% reduction, p = 0.06) when compared to vehicle, however these changes did not reach statistical significance. The combination of CB-839 and TMZ more significantly reduced both tumor glutamate (30% reduction, p = 0.03) and aspartate (34% reduction, p &amp;lt; 0.001) when compared to TMZ monotherapy. CB-839 did not significantly increase DNA damage compared to vehicle, and the combination of CB-839 and TMZ did not significantly increase DNA damage compared to TMZ monotherapy. The mean tumor: plasma ratios of CB-839 concentrations were 0.68 and 0.58 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively. The mean brain: plasma ratios of CB-839 concentrations were 0.07 and 0.12 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively. Conclusion: The addition of CB-839 to TMZ significantly reduces glioma aspartate and glutamate in an IDH1 mutant PDX glioma model, without any impact on DNA damage. Survival studies are in progress to assess the efficacy of CB-839 when used in combination with RT and/or TMZ. Citation Format: Sani Haider Kizilbash, Danielle M. Burgenske, Samuel McBrayer, Sandhya Devarajan, Shiv K. Gupta, Taro Hitosugi, Lihong He, Mark A. Schroeder, Brett L. Carlson, Marina Gelman, Charles A. Kunos, Joel M. Reid, Alex A. Adjei, Jann N. Sarkaria. The addition of CB-839 to temozolomide significantly reduces glioma aspartate and glutamate in an IDH mutated patient derived glioma xenograft 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 3870.