Estrogen Pellets Research Articles

GPER expression prevents estrogen-induced urinary retention in obese mice

Long-term administration of exogenous estrogen is known to cause urinary retention and marked, often fatal, bladder distention in both male and female mice. Estrogen-treated mice have increased bladder pressure and decreased urine flow, suggesting that urinary retention in estrogen-treated mice is due to infravesicular obstruction to urine outflow. Thus, the condition is commonly referred to as bladder outlet obstruction (BOO). Obesity can also lead to urinary retention. As the effects of estrogen are mediated by multiple receptors, including estrogen receptors ERα and ERβ and the G protein-coupled estrogen receptor (GPER), we sought to determine whether GPER plays a role in estrogen-induced BOO, particularly in the context of obesity. Wild type and GPER knockout (KO) mice fed a high-fat diet were ovariectomized or left ovary-intact (sham surgery) and supplemented with slow-release estrogen or vehicle-only pellets. Supplementing both GPER KO and wild type obese mice with estrogen for 8 weeks resulted in weight loss, splenic enlargement, and thymic atrophy, as expected. However, estrogen-treated obese GPER KO mice developed abdominal distension, debilitation, and ulceration of the skin surrounding the urogenital opening. At necropsy, these mice had prominently distended bladders and hydronephrosis. In contrast, estrogen-treated obese wild type mice only rarely displayed these signs. Our results suggest that, under conditions of obesity, estrogen induces BOO as a result of ERα-driven pathways and that GPER expression is protective against BOO.

Abstract PO1-26-05: Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations

Abstract Background: CAR T cells for the treatment of solid tumor cancers has not yet achieved the same success as CAR T cells for treatment of blood cancers. Two of the hurdles that must be cleared for effective use of CAR T cells for solid tumor cancers are: 1) CAR T cell exhaustion; and 2) failure to recognize and kill low antigen expressing cancer cells. A promising approach to overcoming CAR T cell exhaustion, referred to as “1XX” was developed in the Sadelain Lab at MSKCC. Mutation of Tyrosines to Phenylalanine in ITAMs 2 and 3, of the CD3z signaling domain, restrict signaling to ITAM 1. This slowing down of signaling has been reported to be effective at eliminating or greatly reducing CAR T cell exhaustion in blood cancers [Feucht, et al 2019; Park et al 2022; Schoutrop, et al 2023]. Purpose: To evaluate the potential of the 1XX mutations to overcome CAR T cell exhaustion and inability to kill low antigen expressing cells in an animal model of breast cancer. Specifically, we tested the ability of three different CARs to eliminate human breast tumors that expressed variable levels of the target antigen, MUC1*, and their ability to suppress tumor recurrence over approximately 100 days. Methods: All three CARs were targeted to the tumor by the same antibody fragment, huMNC2, that recognizes MUC1*, which is the transmembrane cleavage product of MUC1 that functions as a potent growth factor receptor. The CARs are: 1) huMNC2-41BB-CD3z; 2) huMNC2-CD28-CD3z; and 3) huMNC2-CD28-1XX. Heterogeneous tumors expressing different levels of target antigen were made as follows. T47D breast cancer cells were engineered to express even more of the target, MUC1*, and were also engineered to fluoresce green. T47D wild type cells were engineered to fluoresce red. Heterogenous tumors consisting of 250,000 cells were implanted sub-cu into female NSG mice bearing 90-day estrogen pellets. The tumors comprised either 70% wild-type/30% overexpressing cells, or 85% wild-type/15% overexpressing cells or 92.5% wild-type/7.5% overexpressing cells. Tumor engraftment was verified by bioluminescence at Day 6 post implantation. There was a total of 150 animals, 5 animals per group. Animals were administered a single dose of CAR T cells into the tail vein on Day 7, wherein the Effector to Target ratio was either 10:1, 5:1 or 1:1. Tumor volume was measured weekly by Luciferase/Luciferin bioluminescence on an IVIS instrument. In addition, the red versus green fluorescence of the tumor was tracked periodically as an indicator of which cells, high or low antigen expressing, were being killed. Between Day 93 and Day 96, animals were sacrificed, cells were recovered from blood and spleen, recovered tumors were weighed, tumor cells dissociated and fluorescent images were captured to determine which cells escaped CAR T cell killing. Conclusions: The CAR with the 1XX mutations in CD3z, huMNC2-CD28-1XX, was much more effective at suppressing breast tumor recurrence than either CAR with wild-type CD3z. At sacrifice, significantly more CAR T cells were recovered from huMNC2-CD28-1XX than from huMNC2-41BB-CD3z or huMNC2-CD28-CD3z. At high dose, the CARs with wild-type CD3z effectively suppressed the high antigen expressing cells, but the recurrent tumors were essentially made up of the low antigen expressing cells that had escaped CAR T cell killing. At low CAR T cell dose, the CARs with wild-type CD3z appeared to become exhausted about 40 days post treatment, when tumors began to recur. Post sacrifice analysis of the recurrent tumors showed that they were made up of both high and low antigen expressing cells. huMNC2-CD28-1XX effectively killed both the high antigen and low antigen expressing cells as evidenced by the live fluorescent imaging and the post-sacrifice analysis of residual tumor. Citation Format: Cynthia Bamdad, Andrew Stewart, Benoit Smagghe, Mark Carter, Danica Walkley, Kevin Yi, Jac-Leen Nash, Michael Nash, Trevor Grant, Gregory Riley. Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-05.

SAT391 Supratherapeutic Estrogen Levels in Transgender Women Likely From Sublingual Estradiol

Abstract Disclosure: E. Jalal: None. C. Baldwin: None. Transgender medicine is typically managed in the outpatient setting. We report a case where the patient’s hospitalization provided unique insight into hormone management to achieve cis-female range estrogen and testosterone levels in a monitored environment.A 38-year-old transgender woman with HIV (on antiretroviral therapy) complicated by neurocognitive disorder, polysubstance use, mood disorder with previous suicidal ideation, and polytrauma (currently wheelchair bound) was admitted for respiratory failure from MRSA pneumonia. She completed 14 days of antibiotics with resolution of acute medical problems, but ability to secure a safe discharge plan prolonged her hospitalization. Endocrinology was consulted to aid in oral estradiol (E2) management.Prior to admission, patient reported taking 10mg of oral E2 daily. While hospitalized, she was given 6mg E2 daily, but requested resumption of her home dose. Initial total estrogen and testosterone levels were 913 pg/mL (56-213 pg/mL) and 105 ng/dL (264-916 ng/dL), respectively. She denied estrogen pellets, implants, or consumption of additional E2 beyond 6mg daily, nor did she have visitors who provided supplemental hormone; however, she admitted that she does not swallow the E2 pills immediately.Endocrinology initiated intramuscular leuprolide to suppress her total testosterone production to the female range of 0-50 ng/dL, allow for decreased dosing of E2 to maintain female range estrogen levels, and minimize venous thromboembolism (VTE) risk given reduced patient mobility. Her E2 was held for 5 days, with a subsequent total estrogen level of 398 pg/mL (56-213 pg/mL). She was restarted on oral E2 2mg, with total estrogen levels of 238 pg/mL and 517 pg/mL (56-213 pg/mL) at three and six days respectively. Oral E2 was again held for an additional four days, leading to total estrogen level of 119 pg/mL (56-213 pg/mL) and total testosterone of 18 ng/dL (264-916 ng/dL). Her final regimen is now oral E2 1mg daily. She did not develop VTE while hospitalized.Research into the pharmacokinetics of oral versus sublingual E2 is scarce. Differences in absorption explain this patient’s supratherapeutic estrogen level of 913 pg/mL and insufficiently suppressed total testosterone of 105 ng/dL. Sublingual E2 achieves higher peak levels but multi-daily dosing is required to fully suppress testosterone production when compared to its oral counterpart, which corresponds to this patient’s initial laboratory findings. Sublingual E2 also has a lower risk of VTE since it avoids first-pass metabolism. Future research should target alternative routes of estrogen therapy that may potentially be safer than current modalities. Presentation: Saturday, June 17, 2023

Steady-state estradiol triggers a unique innate immune response to allergen resulting in increased airway resistance

RationaleAsthma is a chronic airway condition that occurs more often in women than men during reproductive years. Population studies have collectively shown that long-term use of oral contraceptives decreased the onset of asthma in women of reproductive age. In the current study, we hypothesized that steady-state levels of estrogen would reduce airway inflammation and airway hyperresponsiveness to methacholine challenge.MethodsOvariectomized BALB/c mice (Ovx) were implanted with subcutaneous hormone pellets (estrogen, OVX-E2) that deliver consistent levels of estrogen [68 ± 2 pg/mL], or placebo pellets (OVX-Placebo), followed by ovalbumin sensitization and challenge. In conjunction with methacholine challenge, immune phenotyping was performed to correlate inflammatory proteins and immune populations with better or worse pulmonary outcomes measured by invasive pulmonary mechanics techniques.ResultsHistologic analysis showed an increase in total cell infiltration and mucus staining around the airways leading to an increased inflammatory score in ovarectomized (OVX) animals with steady-state estrogen pellets (OVX-E2-OVA) as compared to other groups including female-sham operated (F-INTACT-OVA) and OVX implanted with a placebo pellet (OVX-Pl-OVA). Airway resistance (Rrs) and lung elastance (Ers) were increased in OVX-E2-OVA in comparison to F-INTACT-OVA following aerosolized intratracheal methacholine challenges. Immune phenotyping revealed that steady-state estrogen reduced CD3+ T cells, CD19+ B cells, ILC2 and eosinophils in the BAL across all experiments. While these commonly described allergic cells were reduced in the BAL, or airways, we found no changes in neutrophils, CD3+ T cells or CD19+ B cells in the remaining lung tissue. Similarly, inflammatory cytokines (IL-5 and IL-13) were also decreased in OVX-E2-OVA-treated animals in comparison to Female-INTACT-OVA mice in the BAL, but in the lung tissue IL-5, IL-13 and IL-33 were comparable in OVX-E2-OVA and F-INTACT OVA mice. ILC2 were sorted from the lungs and stimulated with exogenous IL-33. These ILC2 had reduced cytokine and chemokine expression when they were isolated from OVX-E2-OVA animals, indicating that steady-state estrogen suppresses IL-33-mediated activation of ILC2.ConclusionsTherapeutically targeting estrogen receptors may have a limiting effect on eosinophils, ILC2 and potentially other immune populations that may improve asthma symptoms in those females that experience perimenstrual worsening of asthma, with the caveat, that long-term use of estrogens or hormone receptor modulators may be detrimental to the lung microenvironment over time.

Abstract P5-05-04: Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes

Abstract MetAP2 inhibitors have shown clinical anti-tumor activity, but CNS side effects and poor drug-like properties have generally limited their development. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) polymer backbone. This conjugation approach is intended to improve safety (i.e., limit CNS penetration) while also improving biodistribution and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 recently completed a phase I safety trial in late-stage cancer patients (NCT02743637) and was well-tolerated with no apparent CNS side effects. In prior models of breast cancer, SDX-7320 significantly inhibited the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (i.e., “metabo-oncology”) and also synergized with the PI3Kα inhibitor alpelisib/Piqray® in ER+/Her2- MCF-7 xenografts. The objectives of these experiments were to determine the effect of SDX-7320 on the growth of Her2+ xenografts alone or in combination with the Akt/mTOR inhibitor capivasertib/AZD-5363 and to determine if SDX-7320 could attenuate hyperglycemia induced by capivasertib/AZD-5363. Female athymic, nude mice (9 weeks of age) were surgically implanted with slow-release estrogen pellets (90-day, 0.72 mg, inter-scapular) and three days later 2 x 107 BT474 cells (in Matrigel/RPMI-1640, 50/50, v/v) were injected into the fourth mammary gland. When the group mean tumor volume exceeded 100 mm3, treatment with test agents commenced. SDX-7320 (sc/q4d, 6, 12 mg/kg) exerted significant, dose-dependent inhibition of BT-474 tumor growth (TGI of 61 and 88% respectively), whereas capivasertib/AZD-5363 (po, qd, 100, 200 mg/kg) exhibited significant anti-tumor activity only at 200 mg/kg (54% TGI). The combination of SDX-7320 (12 mg/kg) plus capivasertib/AZD-5363 (200 mg/kg) produced additive effects in that a greater number of mice had tumor regression relative to mice treated with either SDX-7320 or capivasertib/AZD-5363 alone. Tumor tissue from a subset of treatment groups (Vehicle, SDX-7320-12 mg/kg, SDX-7320-12 mg/kg + AZD-5363-200 mg/kg, and AZD-5363-200 mg/kg) was snap frozen and stored at -80oC. PolyA+ RNA was isolated, cDNA libraries were constructed and RNASeq was conducted (range of 20-30 reads per million (RPM) base pairs). Analysis of RNASeq data showed that SDX-7320 alone significantly attenuated the expression of hypoxia-induced genes as well as genes related to the innate immune system. Based on these results, we conclude that SDX-7320 affected tumor growth by limiting the ability of tumors to adapt to a hypoxic microenvironment and by altering the innate tumor-immune microenvironment. In normal male, C57Bl/6 mice, capivasertib/AZD-5363 (200 mg/kg, po) elevated blood glucose after a single dose. Pretreatment with either one dose (24 hours before the Akt/mTOR inhibitor) or multiple doses (Q4D beginning 14 days prior to Akt/mTOR inhibitor) of SDX-7320 (8 mg/kg) significantly inhibited hyperglycemia induced by capivasertib/AZD-5363. In summary SDX-7320 significantly inhibited the growth of BT-474 xenografts, alone and in combination with capivasertib/AZD-5363, which was associated with suppression of hypoxia-induced genes, as well as genes of the innate immune system. In addition, SDX-7320 improved the safety profile of capivasertib/AZD-5363 by attenuating Akt/mTOR inhibitor-induced hyperglycemia, providing further support for the clinical exploration of SDX-7320 in combination with Akt/mTOR inhibitors in Her2+ breast cancer. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Mark Kalinich, Jonathan Wang, Sara Little, Bradley Carver, James Shanahan. Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes [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 P5-05-04.

Abstract 1068: SDX-7320, a novel inhibitor of methionine aminopeptidase 2 (MetAP2), inhibits MCF-7 tumor growth in combination with palbociclib (Ibrance®)

Abstract Small molecule MetAP2 inhibitor TNP-470 previously showed clinical anti-tumor activity, however CNS side effects and poor drug-like properties limited its development. SDX-7320 is a polymer-drug conjugate of a novel MetAP2 inhibitor (SDX-7539), intended to improve biodistribution (limit CNS penetration) and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 has recently completed a phase I trial in late-stage cancer patients (NCT02743637). SDX-7320 was previously shown to inhibit the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (“metabo-oncology”). In addition SDX-7320 synergized with the PI3Kα inhibitor alpelisib (Piqray®) to block the growth of ER+/Her2- MCF-7 xenografts. Here, we show that SDX-7320 inhibits the growth of MCF-7 xenografts alone and in combination with the CDK4/6 inhibitor Palbociclib (Ibrance®). Nude mice were implanted with slow release estrogen pellets and then injected with MCF-7 cells in the fourth mammary gland. Treatment began (n=10/group) when tumors exceeded 100 mm³: SDX-7320 (sc/q4d, 8 mg/kg), palbociclib (po, qd, 20 or 40 mg/kg), SDX-7320 plus palbociclib (20 mg/kg), SDX-7320 plus palbociclib (40 mg/kg). MCF-7 tumor growth inhibition at day 31 relative to vehicle-treated mice: SDX-7320, 29% (NS); palbociclib, 24% and 54% at 20 and 40 mg/kg respectively (NS); SDX-7320 plus palbociclib (20 mg/kg), 64% (p<0.05); and SDX-7320 plus palbociclib (40 mg/kg), 82%, (p<0.01). Tumor samples were homogenized in RIPA buffer containing protease and phosphatase inhibitors for analysis of proteins (by WES). Cell-cycle proteins (cyclin E1, E2, cdk2, cdk4) were decreased in tumor tissue from SDX-7320-treated mice relative to vehicle, and were typically lower in tumors from mice treated with SDX-7320 plus palbociclib (40 mg/kg) compared to either vehicle-treated or SDX-7320-treated mice. A key growth factor-signaling protein Akt was reduced in SDX-7320-treated groups (alone and in combination) as was estrogen receptor alpha (ERα). pAkt (S473) was lowest in SDX-7320-treated tumors and in tumors treated with SDX-7320 plus palbociclib (40 mg/kg). The autophagy marker LC3B was elevated in response to palbociclib (20 and 40 mg/kg) but in combination with SDX-7320, the increase was significantly attenuated. Intracellular pathways and proteins linked to emergence of resistance to palbociclib include cell cycle (cyclin E1), growth factor and hormone signaling (Akt, ERα) and autophagy (LC3B). The protein changes observed (representative of these pathways) suggest that combining SDX-7320 with palbociclib (Ibrance®) may extend the period of progression-free survival in patients with ER+/Her2- breast cancer relative to treatment with palbociclib (Ibrance®) alone. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Sara Little, Andrew Slee, Raphael Nir, Adam Nir, Bradley J. Carver, James Shanahan. SDX-7320, a novel inhibitor of methionine aminopeptidase 2 (MetAP2), inhibits MCF-7 tumor growth in combination with palbociclib (Ibrance®) [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 1068.

Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.

Abstract PO040: Chemoprevention and regression of estrogen-induced atypical endometrial hyperplasia by oral SHetA2 in a rat model

Abstract Introduction: The purpose was to evaluate the chemoprevention efficacy of SHetA2 (NSC 726189) in a rat model of atypical endometrial hyperplasia (AEH). Methods: Female Sprague Dawley rats were ovariectomized, rested for 2 weeks, and subcutaneously implanted with placebo or slow-release estrogen (E2; 1.5 mg/90-day) pellets on the lateral side of the neck. The rats were randomly divided for two studies: 1) AEH prevention, in which SHetA2 treatment was started one day after E2 pellet implantation; 2) AEH regression, in which SHetA2 treatment was started after AEH development (12 weeks after E2 pellet implantation) and continued for 4 weeks. SHetA2 was orally given as a suspension at three different doses of 30, 60 or 90 mg/kg/day. The suspension was prepared by first micronizing SHetA2 powder to < 63 µm and then ultrahomogenizing it in aqueous 30% Kolliphor HS15 until the size of drug particles in suspension was < 20 µm. The presence of AEH in H&E stained uterine sections was evaluated by two pathologists as the study endpoint by the presence of abnormal cells (nuclear enlargement, nuclear membrane irregularities, prominent nucleoli, nuclear hyperchromasia, nuclear pleomorphism, mitotic activity and/or loss of axial polarity) within tissue areas that exhibit at least one of the following features: increased gland to stroma ratio of >1:1, glandular complexity including irregular contours, and cribriform/papillary configurations. Results: E2-treated animals had reduced body weight gain compared to placebo-implanted animals (Repeated Measures ANOVA, p<0.0001). There were no effects of SHetA2 on rat body weights. In the prevention study, uterine to body weight ratios were significantly higher in E2-treated animals compared to placebo (Repeated Measures ANOVA, p<0.0001) and significantly reduced by 30 and 60 mg/kg/day SHetA2 doses (Repeated Measures ANOVA, p=0.0193 and 0.0108, respectively). Also, SHetA2 caused a dose-responsive decrease in AEH incidence in E2-implanted animals from 86.7% (13/15) in the untreated control to 53.3% (8/15), 26.7% (4/15) and 26.7% (4/15) in the 30, 60 and 90 mg/kg/day SHetA2 treatment groups, respectively (Chai Square Analysis p<0.0022). In the regression study, there were no significant differences in uterine to body weight ratios across all groups. Also, SHetA2 caused dose-responsive regression of AEH to normal histology in E2-implanted animals observed by lack of AEH in 18.2% (2/11) of the untreated control group and 83.3% (10/12), 81.8% (9/11) and 75% (9/12) in the 30, 60 and 90 mg/kg/day SHetA2 treatment groups, respectively (Chai Square Analysis p<0.0025). Conclusions: AEH can be induced by implantation of slow-release E2 pellets in ovariectomized rats. SHetA2 significantly prevented AEH development and caused AEH regression to normal endometrium histology in a dose-dependent manner. Further study is warranted to explore the mechanistic chemoprevention action of SHetA2 and translate these findings toward clinical trials. Funding: NCI PREVENT Program Contract HHSN26100008. Citation Format: Vishal Chandra, Rajani Rai, Stan S. Lightfoot, Manolya Kukut Hatipoglu, Lucila Garcia-Contreras, Doris M. Benbrook. Chemoprevention and regression of estrogen-induced atypical endometrial hyperplasia by oral SHetA2 in a rat model [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO040.

Abstract 3687: Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells

Abstract ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic surface antigen that is expressed on breast cancer, but not on normal postpartum tissues. We found ROR1 was a receptor for Wnt5a, and high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). More recently, ROR1 could activate RhoGTPases and enhance tumor growth in breast cancer (Zhang S., et al., PNAS, 116(4), 2019). Such effects of Wnt5a on breast cancer patient-derived-xenograft (PDX) tumors could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. However, the molecular mechanism(s) for how ROR1 contributes such functions remained elusive. We performed mass spectrometry-based proteomic analysis on anti-ROR1 immune precipitates from breast cancer PDX tumors and detected DOCK1 in addition to ROR1, implying that ROR1 associates with DOCK1. DOCK1 (Dedicator of cytokinesis 1, also known as DOCK180) that expressed in breast cancer PDXs and cell lines, is a cytoplasmic protein, and a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1. Moreover, DOCK1 consists SH3 domain, which allows it to bind characteristic motifs (-P-X-X-P-), which often are found in the proline rich domains (PRD) of other proteins. Immunoblot analysis confirmed that ROR1 associated with DOCK1 in breast cancer PDX tumors or MCF7 cells transfected to express ROR1 (MCF7-ROR1) in response to Wnt5a, leading to enhanced activation of Rac1. To explore functional role, we performed a 2-week colony-formation assay using MCF7-ROR1 cells, and found that treatment of Wnt5a could enhance number of colonies of MCF7-ROR1 cells and that such colony formation capability could be inhibited by reducing expression of DOCK1 with specific siRNA, but not by control siRNA, suggesting a dependency on DOCK1 for Wnt5a-enhanced MCF7-ROR1 cells colony formation capability. Moreover, such effects of Wnt5a to enhance colony formation ability of MCF7-ROR1 cells could be inhibit by cirmtuzumab. We also introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1 proline-rich-domain (PRD) at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P⇒A mutants, ROR1P(808)A was unable to recruit DOCK1 to ROR1, or activate Rac1 in response to Wnt5a. Next we examined the tumor-growth potential of MCF7-Ctrl, MCF7-ROR1, or MCF7-P808A cells. The cells (5 × 104 cells/inoculate in RPMI 1640 together with an equal volume of Matrigel) were injected subcutaneously into the second right mammary pad of 4-to 6-week-old Rag2−/−γc−/- mice pretreated with estrogen pellets (17β-estradiol), which were placed subcutaneously in the intrascapular region one week before the cells inoculation. We found that MCF7-ROR1 cells (n=6) had significantly higher tumor growth relative to MCF7 cells (n=8), which had the same tumor growth as that of MCF7-P808A cells (n=6). Moreover, MCF7-ROR1P808A tumors had less activation of Rac1 compared to MCF7-ROR1 cells expressing wild-type ROR1. This study reveals that the recruitment of DOCK1 to ROR1 may be critical for the capacity of Wnt5a to enhance breast cancer tumor growth. Citation Format: Md Kamrul Hasan, George F. Widhopf II, Suping Zhang, Sharon M. Lam, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells [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 3687.

Abstract 27: Development of a rat model of atypical endometrial hyperplasia and a vaginal suppository formulation of SHetA2 for chemoprevention studies

Abstract Introduction: The purposes were to develop a rat model of atypical endometrial hyperplasia and a vaginal suppository of the drug SHetA2 (NSC 726189) for use in a chemoprevention study. Methods: Female Sprague Dawley rats (150-250 g) were ovariectomized to prevent production of endogenous estrogen and progesterone. After a 2 wks rest, placebo or slow release estrogen (E2) pellets were implanted subcutaneously on the lateral side of the neck in the ovariectomized rats randomly assigned to 3 groups (N=25): 1) placebo, 2) 0.5 mg E2/90-day, 3) 1.5 mg E2/90-day. Pellets were replaced after ~10 wks. Rats were weighed wkly. Three rats in each group were sacrificed at 2, 4, 6, 8, 10, 12, 14, and 16 wks after pellet implantation. Organs were collected and weighed. The dimensions of the vaginal canals of six rats were measured to design an aluminum mold to prepare vaginal suppositories containing SHetA2. The mold was custom-made to have a conical shape with a 5.5 mm diameter base (average vaginal canal diameter) and 5 mm height (half of the total length of the vaginal canal - 10.10 mm). A pilot batch of 20 SHetA2 suppositories was prepared using a cocoa butter base, stored at 4°C in an air-tight and light-tight container and evaluated for SHetA2 stability at 1, 2 and 3 months. Results: Rats with placebo implants exhibited continuous body weight gain. In comparison, both E2-treated groups had reduced weight gain (Repeated Measures ANOVA, p<0.0001). At each time point, average uterine horn weights from the E2-treated groups were significantly higher than the placebo group (Repeated Measures ANOVA, p<0.0001). Placebo group rats had normal endometrium with occasional glandular crowding. At 8-10 wks, focal atypia was observed in 1 of 6 rats in the 0.5 mg E2 group and in 4 of 6 rats in the 1.5 mg E2 group. At the 12-wk time point, atypical hyperplasia was observed in 7 of 9 rats in the 0.5 mg E2 group and in 8 out of 9 rats in the 1.5 g E2 group. Repeated measures ANOVA of organ/body weight ratios indicated that the E2-treated animals had significantly lower spleen weights (p=0.0019) and higher liver (p=0.0001), kidney (p<0.0001) and pancreas (p=0.0004) weights. The reduced organ weights may be due to the significant reduction in body weight in the E2-treated rats. The SHetA2 suppositories were stable at -20°C for 3 months. Storage of individual suppositories in wells of 48-well plates prevented the twining together that occurred when stored in bulk, and allowed removal from packaging without damaging the suppositories. Conclusions: A 1.5 mg slow release E2 pellet in ovariectomized rats caused development of atypical endometrial hyperplasia within a sufficient timeframe and incidence to allow a chemoprevention study. The mold was suitable to prepare rat-sized suppositories and the resulting SHetA2 suppositories can be safely stored for use within 3 months. Funding: NCI PREVENT Program Contract HHSN26100008 Citation Format: Vishal Chandra, Rajani Rai, Sanam Hussain, Lucila Garcia, Manolya Hatipoglu, Doris Benbrook. Development of a rat model of atypical endometrial hyperplasia and a vaginal suppository formulation of SHetA2 for chemoprevention studies [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 27.

Abstract P3-11-13: Synergistic inhibition of MCF-7 mammary gland tumor growth with Piqray® (alpelisib) plus SDX-7320, a novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor

Abstract Mutations in the PI3K pathway have been observed in about 40% of ER+/Her2- breast cancer patients. Amplification as well as mutation of the catalytic p110α subunit of PI3K results in activation of this pathway within tumors, conferring a growth advantage due to increased transmission of intracellular growth signals. Recently a selective inhibitor of p110α, Piqray®(alpelisib) has been approved by the FDA (in combination with the estrogen receptor degrader/antagonist Faslodex/fulvestrant) for the treatment of ER+/Her2- breast cancers harboring mutation(s) in the p110α subunit of PI3K. Small molecule MetAP2 inhibitors have previously shown clinical anti-tumor activity. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone. This is intended to alter biodistribution (limit CNS penetration) and improve pharmacokinetics relative to small molecule, fumagillin-derived MetAP2 inhibitors. SDX-7320 has completed a phase I trial in late-stage cancer patients (NCT02743637). The objective of this study was to evaluate the anti-tumor efficacy of Piqray®(alpelisib) in combination with SDX-7320 in a model of PI3K-mutated, ER+ breast cancer (i.e., MCF-7). Female nude mice had estrogen pellets surgically implanted and after two weeks of recovery, MCF-7 cells were injected into the fourth mammary gland. When tumors became palpable (i.e., > 50 mm3) treatment with SDX-7320 (dosed subcutaneously Q4D at 8 or 16 mg/kg) and/or Piqray®(alpelisib) (dosed PO, QD at 25 or 45 mg/kg) commenced. Combinations included SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 25 mg/kg as well as SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 45 mg/kg. Endpoints included tumor volume, body weight, terminal plasma glucose, insulin, leptin and adiponectin. Analysis of tumor tissue for molecular endpoints of PI3K pathway activation were also investigated (e.g., S6, Akt, 4E-BP1). Anti-tumor efficacy was assessed by calculating the %change in tumor volume from baseline for each individual animal, then comparing the average change for each group at the end of the study (day 64). One-way ANOVA with multiple comparisons was conducted to determine significance of differences in final tumor volume on day 64 relative to vehicle. Treatment with SDX-7320 at 8 or 16 mg/kg inhibited MCF-7 tumor growth -19% and -17% relative to vehicle at day 64 (NS), respectively. Piqray®(alpelisib) inhibited tumor growth in a dose-dependent manner (-30% (NS) and -82% (p<0.05) for 25 and 45 mg/kg, respectively). The combination of SDX-7320 at 8 mg/kg with Piqray®(alpelisib) at 25 mg/kg resulted in synergistic efficacy, with -105% inhibition of tumor growth relative to vehicle at day 64 (p<0.01).The combination of SDX-7320 at 8 mg/kg with Piqray®(alpelisib) at 45 mg/kg resulted in an additive effect on tumor growth relative to vehicle (-100%; p<0.01). These results show that SDX-7320 (8 mg/kg) is synergistic with Piqray®(alpelisib; 25 mg/kg) in control of orthotopic MCF-7 mammary gland tumors. A clinical trial combining SDX-7320 with Piqray®(alpelisib) plus fulvestrant is planned to start in Q1/2020 and will assess the ability of SDX-7320 to enhance the efficacy of Piqray®(alpelisib) in treating PI3K-mutated, ER+/Her2- breast cancer. Citation Format: Peter Cornelius, Benjamin Mayes, Sara Little, Andrew Slee, Adam Nir, Raphael Nir, Bradley Carver, James Shanahan. Synergistic inhibition of MCF-7 mammary gland tumor growth with Piqray® (alpelisib) plus SDX-7320, a novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor [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 P3-11-13.

Estrogen modulation of pain perception with a novel 17\u03b2-estradiol pretreatment regime in ovariectomized rats

Estrogen plays substantial roles in pain modulation; however, studies concerning sex hormones and nociception often yield confusing results. The discrepancy could be a result of lack of consensus to regard estrogen as a variable when working with animal models; thus, the influence of hormones’ fluctuations on nociception has continually been neglected. In the present study, we designed a novel hormone substitution model to aid us to evaluate the effects of estrogen’s long-term alterations on ovariectomy (OVX)-induced mechanical hyperalgesia and the expression of estrogen receptors(ERs). OVX rats were implanted with slow-release estrogen pellets at differently arranged time points and doses, such that a gradual elevation or decrease of serum estrogen levels following a relatively stable period of estrogen replacement was achieved in rats. Our results demonstrated that gradual estrogen depletion rather than elevation following the stable period of estrogen substitution in OVX rats alleviated OVX-induced mechanical hyperalgesia in a dose-independent manner, and the opposite estrogen increase or decrease paradigms differently regulate the expression of spinal ERs. Specifically, in rats rendered to continuously increased serum estrogen, the early phase estrogen-induced anti-nociception effect in OVX rats was eliminated, which was accompanied by an over-activation of ERα and a strong depression of ERβ, while in the OVX rats subject to gradual decrease of estrogen replacement, both ERα and ERβ increased modestly compared with the OVX group. Thus, the present study demonstrated that estrogen increase or decrease modulate nociception differently through change of spinal ERs.

Sex-dependent effects of estrogen pellets in human liver cancer xenograft models.

Liver cancer shows noticeable differences in the incidence rate and mortality between genders. To investigate the estrogen effect on tumor progression in liver cancer, we developed a xenograft model using estrogen pellets. SK-Hep1 cells (human male liver carcinoma) were inoculated into male or female nude mice. Subsequently, estrogen pellets were subcutaneously implanted into these xenograft models. Interestingly, the marked adverse effect of estrogen pellets (0.5mg/21days) were observed in the male-derived xenograft model, with increased ulcerative dermatitis in male mice than in female mice. Additionally, necrosis was observed in male mice with SK-Hep1-derived tumors. However, the estrogen pellet (0.5mg/60days) did not exhibit these adverse effects. Tumor growth in female mice was significantly suppressed by estrogen (0.5mg/60days). Tumor growth was also suppressed in male mice implanted with estrogen (0.5mg/60days), but the suppression was not significant. We found that estrogen-induced skin damage was more severe in male mice than female mice. The tumor suppression of estrogen was effective in female mice compared to male mice bearing liver cancer. The results suggest that the sex difference affects estrogen activity and thus should be considered in the preclinical assessment.

Translin restricts the growth of pubertal mammary epithelial cells estrogen-independently in mice

Translin, a ubiquitous RNA/DNA-binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses endoribonuclease activity and plays a physiological role in restricting the size and differentiation of mesenchymal precursor cells. However, the precise role of Translin in epithelial cells remains unclear. Here, we show evidence that Translin restricts the growth of pubertal mammary epithelial cells. The mammary epithelia of Translin-null females exhibited retarded growth before puberty, but highly enhanced growth and DNA synthesis with increased ramification after the onset of puberty. Primary cultures of Translin-null mammary epithelial cells showed augmented DNA synthesis in a ligand-independent and ligand-enhanced manner. Translin-null ovariectomized mice implanted with slow-release estrogen pellets showed enhanced length and ramification of the mammary glands. Mammary epithelial growth was also observed in ovariectomized Translin-null mice implanted with placebo pellets. Luciferase reporter assays using embryonic fibroblasts from Translin-null mice showed unaltered estrogen receptor α function. These results indicate that Translin plays a physiological role in restricting intrinsic growth, beyond mesenchymal cells, of pubertal mammary epithelial cells.

Hypothalamic-pituitary-adrenal axis responsivity to an acute novel stress in female rats subjected to the chronic mild stress paradigm

The chronic mild stress (CMS) paradigm is the most frequently investigated animal model for major depression. The hypothalamic-pituitary-adrenal (HPA) axis participates in the generation of depressive symptomatology. We examined whether the depression-like state induced by CMS is associated with immediate changes in HPA axis activation in response to a novel acute stress and whether this response could be modified by hormonal status. Adult female Wistar rats were ovariectomized and received estrogen or vehicle pellets. After 2 weeks, rats were subjected to CMS (or control) conditions for 2.5 or 4.5 weeks. Rats were subsequently subjected to restraint stress for 1 h, and plasma corticosterone (CT) levels were determined before (2:00 p.m.) and after acute stress induction (3:00 and 4:00 p.m.). CT levels and FOS expression were measured in the medial parvocellular subdivision of the PVN (PaMP), central (CeA) and medial amygdala (MeA) and ventral subiculum of the hippocampus (vSub). Plasma CT levels in animals treated with 6.5 weeks of estrogen were elevated before and 1 h after restraint stress induction. Results indicate that the estrogen chronicity and CMS exposure impacted CT secretion. Neuronal PaMP, CeA, MeA and vSub activity decreased after 4.5 weeks of CMS in all groups. No differences were detected between CMS and non-CMS groups. These data suggest that the HPA central hyporesponsiveness observed in the experimental groups subjected to a longer protocol period was independent to CMS paradigm and estrogen treatment restored partially its activity. These data suggest that additional stressors could be responsible for the observed alterations of the HPA axis.

Abstract 2356: Estrogen medicates sex specific function of epithelial STAT3 in K-ras mutant lung tumorigenesis by reprogramming lung tumor microenvironment

Abstract Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer but have not yielded to therapeutic attack so far. We have recently shown a sex-specific role for epithelial Stat3 signaling in the pathogenesis of K-ras mutant lung cancer. We specifically found that deletion of STAT3 in K-ras mutant lung epithelial cells (LR/STAT3Δ/Δ mice) significantly reduced lung tumor burden in female mice which was associated with an enhanced anti-tumor immune response but, surprisingly, caused a dramatic enhancement of lung tumorigenesis and induction of a NF-κB driven pro-tumor immune response in male mice. To further dissect the mechanism of STAT3 dependent sex disparity in the pathogenies of K-ras mutant lung cancer, we performed loss- and gain- of function studies to manipulate estrogen signaling in both female and male mice. In LR/Stat3Δ/Δ males, implantation of estrogen pellet led to significantly reduced lung tumor burden and decreased neutrophils in the lung. This was accompanied by decreased Il6, and Cxcl1 expression explaining reduced neutrophil numbers. We also detected reduced expression of immunosuppressive markers; Arg1, Nos2, Tgfb, Il10, indicating to an attenuated immunosuppressive pro-tumor microenvironment. On the other hand, bilateral oophorectomy in female LR/Stat3Δ/Δ mice led to higher tumor burden, higher lung neutrophil counts, and increased Il6, and Cxcl1 expression. We also found decreased expression of Gzmb, and Ifng, and increased expression of T regulatory markers Foxp3 and Il10, suggesting an attenuated cytotoxic immune response but a stronger immune-inhibitory response. Hormonal replacement therapy by implanting estrogen pellet in oophorectomized LR/Stat3Δ/Δ females rescued the tumor-promoting effect of estrogen depletion characterized by reduced tumor number and neutrophil infiltration, decreased expression of Il6, Cxcl1, Foxp3 and Il10, and increased Gzmb and Ifng expression. Taken together, our study suggests that there is a differential regulation of NF-κB activation in K-ras mutant lung epithelium within sex, and estrogen plays a protective role (anti-tumor) by suppressing the activation of NF-κB pathway and reformatting the lung tumor microenvironment toward an anti-tumor phenotype. Our finding could lead to development of personalized (e.g. sex-based) immunotherapeutic and/or preventive modalities for K-ras mutant lung cancer. Citation Format: Shanshan Deng, Marco Ramos-Castaneda, Walter Velasco Torrez, Oscar Noble, Neha Daga, Mauricio Caetano, Seyed Javad Moghaddam. Estrogen medicates sex specific function of epithelial STAT3 in K-ras mutant lung tumorigenesis by reprogramming lung tumor microenvironment [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 2356.

Abstract P2-02-03: FASN inhibition as a potential treatment for therapy of endocrine resistant breast cancer

Abstract INTRODUCTION: Fatty acid synthase (FASN) is a key enzyme in tumor cell biology controlling endogenous lipid biosynthesis. It is overexpressed in a biologically aggressive subset of tumors, including breast carcinoma. We previously reported prolonged stabilization of disease with TVB-2640 in patients with advanced metastatic breast cancer, including some endocrine resistant ER+ tumors. Using in vitro and in vivo models, we assessed the role of FASN inhibition by TVB-3166 (preclinical version of TVB-2640) for treatment of endocrine resistant breast cancer. METHODS: Breast tumor cells were incubated with TVB-3166 (200nM), imaged and analyzed by automated Live-Cell analysis system (IncuCyte). For tumor growth inhibition, cells (2X106)were subcutaneously injected into SCID mice implanted with estrogen pellets. Once tumors were measurable, mice were divided into treatment groups: tamoxifen (4mg/kg), TVB-3166 (60mg/kg) and the combination. Patient tumor explants were incubated for 72h on gelatin sponges in culture medium in the absence or presence of 200nM TVB-3166. Tissue were fixed in 10% formalin and processed into paraffin blocks. Sections were stained with H&E, ERα and Ki67. RESULTS: The effectiveness of FASN inhibition on the growth of tumor cells has been confirmed in a number of breast cancer cell lines such as MCF7, ZR75, MDA-MB-231 and others. TVB-3166 leads to a marked inhibition of growth in tamoxifen resistant (TamR) cells, which 15% greater than in the parent line. IHC and Western blot showed FASN inhibition leads to significantly reduction of ERα levels. Immunofluorescent confocal microscopy showed inhibition of FASN by TVB-3166 alters subcellular localization of ERα. TVB-3166 was able to significantly inhibit tamoxifen resistant breast tumor growth in mice (p<0.05). Additionally, TVB-3166 treatment of primary tumor explants decreased their proliferation (Ki67) compared to untreated controls (21% vs 38%, p<0.01). CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treating of endocrine resistant breast cancer. RNA sequencing of tumor explants is being performed to evaluate FASN inhibition impact on canonical and non-canonical ERα signaling pathways. Citation Format: Gruslova A, Sareddy GR, Vadlamudi RK, Viswanadhapalli S, Brenner A. FASN inhibition as a potential treatment for therapy of endocrine resistant breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-03.

Abstract A58: Application of evolutionary principles to control ER+ breast tumors

Abstract Introduction: Estrogen receptor-positive (ER+) breast cancers are initially treated with estrogen deprivation, usually using estrogen receptor modulator drugs (SERM) that are administered at near maximal dose until progression. However, in many of ER+ tumors, preexisting resistant populations that do not express ER on immunohistochemistry are observed. Furthermore, a number of other resistance mechanisms to SERM drugs have been observed including overexpression of MDR1 cell-membrane systems, which consume energy to extrude the drug from the cell. While the vast majority of ER+ tumors respond to SERM drugs, resistant populations can rapidly emerge due to preexisting or acquired phenotypic strategies. Herein, we examine nonconventional treatment strategies, based on the Darwinian evolutionary dynamics that govern tumor evolution, designed to maintain tumor control while delaying evolution of resistance. Materials and Methods: Different cohorts (20, 24, 4, and 41) of Nu/nu mice were injected in the mammary fat pad with 5*106 MCF7 cells. Prior to the cell injections, an estrogen pellet was subcutaneously implanted in mice. When tumors reached 300 mm3, mice were randomly distributed in treatment groups using different algorithm that combined tamoxifen, paclitaxel, and periods of no treatment. Tumor volumes were monitored by MRI, using T2-weighted images. At the end of the experimental time, tumors were collected and stained for IHC studies with H&E, CD31, SMA, MDR1, and ER. Results: Treatments in which tamoxifen therapy was interrupted by none-drug periods permitted tumor control equal to that of high-dose standard treatment while maintaining a large population of ER+ cells. Algorithms in which tamoxifen and paclitaxel were combined achieved tumor control equal to standard therapy and decreased MDR1 expressions. Conclusions: Treatment algorithms designed to exploit evolutionary principles to delay or prevent emergence of resistant populations maintained tumor control using lower cumulative drug doses than the standard-of-care therapy while maintaining populations of cells that are sensitive to the therapy. Note: This abstract was not presented at the conference. Citation Format: Pedro M. Enriquez-Navas, Libia Garcia, Robert J. Gillies, Robert A. Gatenby. Application of evolutionary principles to control ER+ breast tumors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A58.

Abstract 2157: Chemotherapeutic tolerability and estrogen dose response in the B6;129-Rag2 tm1Fwa IL2rg tm1Rsky/DwlHsd (R2G2) mouse model

Abstract The B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA-damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole-body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA-damaging oncology drugs including 5-fluorouracil (5-FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n = 10 per group). 5-FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models. Results show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous estrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous estrogen pellets, developing negative secondary effects resulting in removal from study. We performed an estrogen pellet dose-response study using four doses of 60-day release 17-β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n = 10 per group). R2G2 mice show dose-dependent effects of estrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model. In conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA-damaging chemotherapies or when estrogen supplementation is required for xenograft growth. Citation Format: Jamie L. Naden, Michele Melton, Athena Bast, Jermaine Gardner. Chemotherapeutic tolerability and estrogen dose response in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2157.

PO-217 Loss of Kmt2c methyltransferase activity contributes to prostate tumorigenesis in a mouse model of prostate cancer

IntroductionThe B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA damaging oncology drugs including 5-fluorouracil (5-FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n=10 per group).Material and methods5-FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models.Results and discussionsResults show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous oestrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous oestrogen pellets, developing negative secondary effects resulting in removal from study. We performed an oestrogen pellet dose response study using four doses of 60 day release 17-β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n=10 per group). R2G2 mice show dose dependent effects of oestrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model.ConclusionIn conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA damaging chemotherapies or when oestrogen supplementation is required for xenograft growth.