Abstract. There is a pressing need to develop animal models as well as treatment appropriate for age-specific radiation injuries. The minipig represents a promising animal model for testing the effects of radiation on the pediatric population. We subjected piglets, age 6 weeks old (corresponding to less than 2 years old in human), to either sham irradiation or to total body irradiation ( 60 Cobalt 0.6 Gy/min) at hematopoietic doses spanning from 1.6 Gy to 2.0 Gy, and determined the dose-survival relationship and course of radiation injury in the presence of minimal supportive care. The LD50/45 was determined to be 1.83 Gy [CI 1.70 -1.91]. The course of hematopoietic acute radiation syndrome (H-ARS) in the piglet model resembled that of humans, with four distinct phases namely, prodromal phase, latent phase, manifest illness phase, and recovery or death. Kinetics of blood cell loss such as sudden lymphopenia, decline in neutrophil counts preceded by initial granulocytosis, erythrocytopenia, and thrombocytopenia with a characteristic shoulder followed by partial recovery mimicked the expected radiationinduced changes. Moribund animals were characterized by anorexia, lethargy, fever or hypothermia, bleeding, and dyspnea. Upon euthanasia, animals displayed dose dependent bone marrow hypoplasia and hemorrhages in several organs. Granulocyte colony stimulating factor (G-CSF), a countermeasure approved for H-ARS in humans and effective in adult minipig, was tested in the piglets. Administration of G-CSF enhanced survival by 37.5% and reduced both duration as well as nadir of neutropenia. In conclusion, the minipig provides a practical and feasible animal model for H-ARS and development of radiation countermeasures for the pediatric population.

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.

In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the PIK3CA, TP53, or E-cadherin genes. Of these co-occurring mutations, HER2 and PIK3CA mutations are the most prevalent gene pair, with approximately 40% of HER2 mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we bred genetically engineered mice with the HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers show accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancers have resistance to the pan-HER tyrosine kinase inhibitor, neratinib, but are effectively treated by neratinib plus trastuzumab deruxtecan. Proteomic and RNA-Seq analysis of HP breast cancers showed increased gene expression of Cyclin D1 and p21WAF1/Cip1 and changes in cell cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for HP breast cancers with neratinib plus palbociclib showing a statistically significant reduction in mouse HP tumors as compared to either drug alone. We validated both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations using a human breast cancer patient-derived xenograft with very similar HER2 and PIK3CA mutations. Further, these two drug combinations effectively treated spontaneous lung metastasis in syngeneic mice transplanted with HP breast cancer organoids. Both of these drug combinations are being tested in phase 1 clinical trials and this study provides valuable preclinical evidence for them.

In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the PIK3CA, TP53, or E-cadherin genes. Of these co-occurring mutations, HER2 and PIK3CA mutations are the most prevalent gene pair, with approximately 40% of HER2 mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we bred genetically engineered mice with the HER2 V777L; PIK3CA H1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using cancer organoids. HP breast cancers show accelerated tumor formation in vivo and increased invasion and migration in in vitro assays. HP breast cancers have resistance to the pan-HER tyrosine kinase inhibitor, neratinib, but are effectively treated by neratinib plus trastuzumab deruxtecan. Proteomic and RNA-Seq analysis of HP breast cancers showed increased gene expression of Cyclin D1 and p21WAF1/Cip1 and changes in cell cycle markers. Combining neratinib with CDK4/6 inhibitors was another effective strategy for HP breast cancers with neratinib plus palbociclib showing a statistically significant reduction in mouse HP tumors as compared to either drug alone. We validated both the neratinib plus trastuzumab deruxtecan and neratinib plus palbociclib combinations using a human breast cancer patient-derived xenograft that has very similar HER2 and PIK3CA mutations. Both of these drug combinations are being tested in phase 1 clinical trials and this study provides valuable preclinical evidence for them.

In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the PIK3CA, TP53, or E-cadherin genes. Of these co-occurring mutations, HER2 and PIK3CA mutations are the most prevalent gene pair, with approximately 40% of HER2 mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we bred genetically engineered mice with the (loxP-STOP-loxP) HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using breast cancer organoids. HP mice rapidly developed invasive mammary adenocarcinoma at a median time of 2.1 weeks after adenoviral Cre injection into the mammary gland. Organoids from these breast cancers showed increased number of buddings in branching morphogenesis assay and increased migration and invasion in vitro. In vivo, HP breast cancers are resistance to the pan-HER tyrosine kinase inhibitor, neratinib, but are effectively treated by the combination of neratinib plus trastuzumab deruxtecan (T-DXd). Ex vivo, we found strong synergy between neratinib and T-DXd in HP organoids. Proteomic and RNA-seq analysis of HP breast cancers showed increased gene expression of CCND1 (cyclin D1) and CDKN1A (which encodes p21WAF1/Cip1) and changes in cell cycle markers. An increase in p-p53, p-p27, and p-PDK1 in HP organoids was seen. The GSEA analysis showed that the mTOR pathway and the MYC target signature were significantly upregulated in the HP organoid group. As p21 stabilizes the cyclin D1-CDK4/6 complex to further activate CDK4/6, we found CDK4/6 inhibitors inhibit cell proliferation in HP mice-derived organoids. Combining neratinib with CDK4/6 inhibitors was another effective strategy for HP breast cancers with neratinib plus palbociclib showing a statistically significant reduction in mouse HP tumors as compared to either drug alone. We validated both the neratinib plus T-DXd and neratinib plus palbociclib combinations using a human breast cancer patient-derived xenograft that has HER2 and PIK3CA mutations very similar to our transgenic mouse. This study provides valuable preclinical evidence for these drug combinations, which are being tested in phase 1 clinical trials. Citation Format: Xiaoqing Cheng, Yirui Sun, Maureen Highkin, Nagalaxmi Vemalapally, Xiaohua Jin, Brandon Zhou, Julie L. Prior, Ashley R. Tipton, Shunqiang Li, Anton Iliuk, Samuel Achilefu, Ian S. Hagemann, John Edwards, Ron Bose. Breast cancer mutations HER2V777L and PIK3CAH1047R activate the p21-CDK4/6 -Cyclin D1 axis driving tumorigenesis and drug resistance. [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 5778.