Patients with hormone receptor-positive (HR+) breast cancers frequently recur with metastatic disease 5-20 years after initial diagnosis and completion of chemotherapy and prolonged hormonal therapy treatment. A potential mechanism of resistance in HR+ patients is the presence of slow dividing or dormant well-differentiated cancer cells that result in clinically detectable metastases after a prolonged latency. One way to eliminate these is immunotherapy. The objective of this study is to target HR+ breast cancers using a natural killer (NK) cell based approach that targets cell surface receptors in HR+ breast cancer. The type I IGF receptor (IGF1R) is expressed on HR+ breast cancers and its expression is tightly related to ER function. We hypothesized that a drug targeting NK cells to IGF1R expressing HR+ breast cancer cells will be effective in killing them. We generated IGF1R specific Tri specific Killer Engagers (TriKE) that leverages expression of IGF1R on HR+ cells to cause NK cell directed cell death. The IGF1R TriKE consists of humanized single domain antibody (VHH) sequence of a CD16 nanobody to engage and activate NK cells, a humanized VHH sequence of a nanobody against IGF1R to target HR+ breast cancer cells, and an IL-15 molecule between them to drive NK cell expansion and survival. The IGF1R TriKE did not bind embryonal fibroblasts from IGF1R knock out mice (R- cells) but bound R-/IGF1R cells (R- cells engineered to express human IGF1R) and to IGF1R+ MCF-7 & T47D breast cancer cells indicating specific binding to IGF1R on cells. It did not cause downregulation of IGF1R levels or inhibit signaling via IGF1R in multiple HR+ breast cancer cells. It specifically induced human NK cell degranulation, measured by surface CD107a expression and intracellular IFNγ production, against IGF1R+ target breast cancer cells (MCF-7, T-47D, ZR-75-1) in a functional assay compared to the anti-IGF1R nanobody. It did not induce degranulation of NK cells against IGF1R negative cells suggesting that the functionality was specific for IGF1R positive targets. The TriKE enhanced cell killing of HR+ breast cancer cells as measured by cytotoxicity assays. We next investigated the effect of IGF1R TriKE on tumor growth of estrogen stimulated proliferating tumors and in a quiescence model of breast cancer. Female NSG mice bearing MCF-7L tumors supplemented with 17-β-estradiol (E2) were randomized to these cohorts: control with no human NK (huNK) cells, huNK cells+IL-15 and huNK cells+TriKE treatments. huNK cells were infused iv weekly and IL-15 or IGF1R TriKE thrice a week for 4 weeks. IGF1R TriKE inhibited estrogen-stimulated tumor growth in 3 independent experiments. Further, CD56+ huNK in blood of mice treated with the TriKE persisted for 3 weeks compared to IL-15 cohort and were also detected in tumor sections of TriKE cohort by huCD45 staining. To test the effect in a model of quiescence, female ovariectomized NSG mice were implanted with MCF-7L cells and given 17-beta-E2. When tumors were 150mm3, E2 was withdrawn to allow cells to become quiescent and two weeks later mice were randomized to same treatment groups as above. IGF1R TriKE significantly killed quiescent tumor cells compared to control and IL-15 groups. Our data suggest that IGF1R TriKE kills both proliferating and quiescent HR+ breast cancers and could be a potential approach to eliminate slow growing cells in HR+ patients. Courtney Baar, Emily Chiu, Yvette Soignier, Jeffrey Miller, Martin Felices, Deepali Sachdev. IGF1R targeted NK cell engager kills proliferating and quiescent hormone receptor positive breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB231.

Abstract Patients with hormone receptor-positive (HR+) breast cancers frequently recur with metastatic disease 5-20 years after the initial diagnosis and completion of chemotherapy and prolonged treatment with hormonal therapy. Thus, patients with metastatic HR+ breast cancer need new therapeutic options. A potential mechanism of therapeutic resistance in HR+ patients is the presence of slow-growing, well-differentiated cancer cells that result in clinically detectable metastases after a prolonged latency. One way to eliminate these cancer cells is with immunotherapy (IT). Unfortunately HR+ breast cancers have not responded to current T-cell based IT. The objective of this study is to target HR+ breast cancers using a natural killer (NK) cell based approach that targets cell surface receptors in HR+ breast cancer. The type I IGF receptor (IGF1R) is expressed on HR+ breast cancers and its expression is tightly related to ER function. Endocrine therapy resistance is associated with increased expression of the A-isoform or fetal form of IR (IR-A) and depend on IR signaling. We hypothesized that a drug that would target NK cells to IGF1R or IR-A expressing HR+ cells will be effective in killing them. We generated IGF1R or IR-A specific Tri specific Killer Engagers (TriKE) that leverage expression of IGF1R and IR-A on HR+ cells to cause NK directed death. The IGF1R TriKE consists of humanized single domain antibody (VHH) sequence of a CD16 nanobody to engage and activate NK cells, a humanized VHH of a nanobody against IGF1R to target HR+ breast cancer cells, and an IL-15 molecule between them to drive NK cell expansion and survival. The IGF1R TriKE did not bind embryonal fibroblasts from IGF1R knock out mice (R- cells) but bound R-/IGF1R cells (R- cells engineered to express human IGF1R) and to IGF1R+ MCF-7 and T47D breast cancer cells indicating specific binding to IGF1R on cells. It did not cause downregulation of IGF1R levels in multiple HR+ breast cancer cells and did not inhibit signaling via IGF1R. It specifically induced NK cell degranulation, measured by surface CD107a expression and intracellular IFNγ, against IGF1R+ target breast cancer cells (MCF-7, T-47D, ZR-75-1) in a functional assay compared to the anti-IGF1R nanobody. It did not induce degranulation of NK cells against IGF1R negative cells suggesting that the functionality was specific for IGF1R expressing targets. The IGF1R TriKE enhanced cell killing of HR+ breast cancer cells as measured by cytotoxicity assays. IGF1R TriKE combined with human NK cells inhibited tumor growth of MCF-7 tumors in NSG mice compared to control groups. Human NK cells persisted in peripheral blood three weeks after final NK infusion based on flow cytometry assessment. The IR-A TriKE incorporated a novel engineered non-immunoglobulin IR-A specific peptide binder. Our preliminary data show that the IR-A TriKE degranulated NK cells against breast cancer cells overexpressing IR-A. These data indicate that this strategy could be an effective treatment for HR+ metastatic breast cancer and make them responsive to IT. Citation Format: Courtney Baar, Emily Chiu, Yvette Soignier, Behiye Kodal, Jeffrey S Miller, Martin Felices, Deepali Sachdev. Trispecific killer engagers against IGF1R and fetal form of insulin receptor induce human natural killer cell mediated killing of hormone receptor positive breast cancer in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B040.

Abstract Disclosure: S.L. Gerhardt: None. C. Baar: None. I. Johnson: None. D. Sachdev: None. The most common subtype of breast cancer is hormone receptor-positive (HR+). While these patients are successfully treated with endocrine therapies (ET), over 30% of patients develop resistance and require other therapies. Cyclin-dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. Three CDK4/6 inhibitors (CDK4/6i), palbociclib, abemaciclib, and ribociclib, have recently been approved in combination with ET. CDK4/6i block phosphorylation of retinoblastoma (Rb) and cause G1/G0 arrest and are standard of care for treating metastatic HR+ Her2- advanced breast cancer with ET. Patients who respond to the CDK4/6i eventually develop resistance to the drugs. Thus, understanding the mechanisms of resistance to CDK4/6i and the role of growth-factor signaling will allow the identification of other pathways that can be targeted. To understand the mechanisms of resistance, we generated HR+ breast cancer cells (MCF-7) with acquired resistance to palbociclib (MCF-7/PalboR) or abemaciclib (MCF-7/AbemaR) by culturing them in increasing concentrations of the drugs. As shown by others, we found that acquired resistance to CDK4/6i results in loss of Rb and thus Rb phosphorylation. We analyzed signaling pathways and in vitro proliferation in the MCF-7/PalboR and MCF-7/AbemaR compared to the CDK4/6i sensitive matched parent cells (MCF-7L MP). MCF-7/PalboR and MCF-7/AbemaR had increased levels of CDK2 and cyclin E2. Both MCF-7/PalboR and MCF-7/AbemaR cells had upregulated type I IGF receptor (IGF1R) and insulin receptor (IR) protein levels compared MCF-7L MP as assayed by western blotting and flow cytometry. MCF-7/PalboR and MCF-7/AbemaR showed higher transcript levels of the fetal form of IR (IR-A) which is not expressed in normal adult tissues. IR-A plays a role in cancer progression and has been implicated in resistance to ET. MCF-7/PalboR and MCF-7/AbemaR had increased sensitivity to signaling by insulin-like growth factor I (IGF-I) and insulin compared to MCF-7 MP, with much lower concentrations of IGF-I and insulin phosphorylating the receptors and activating downstream MAPK and PI3K/Akt/mTOR pathways. Further, IGF-I and insulin mediated growth was enhanced to a statistically greater extent in MCF-7/PalboR and MCF-7/AbemaR compared to MCF-7 MP, and this enhanced growth was not blocked by 1200 nM of palbociclib or abemaciclib. MCF-7/PalboR cells were cross resistant to abemaciclib and MCF-7/AbemaR to palbociclib. Combined treatment with CDK4/6i and rapamycin, an mTOR pathway inhibitor, was not sufficient to overcome growth-factor mediated proliferation in MCF-7/PalboR and MCF-7/AbemaR but was in MCF-7 MP. These data indicate that acquired resistance to CDK4/6i is mediated through upregulated IGF1R and IR signaling. Our data suggest that combining CDK4/6i with drugs targeting IGF1R, IR-A, and mTOR signaling should be tested to delay resistance to CDK4/6i. Presentation: Thursday, June 15, 2023

Study Objective To demonstrate the robotic-assisted laparoscopic surgical technique in the resection of an advanced cornual ectopic pregnancy in a morbidly obese patient. Design Video recording of the cornual ectopic resection edited to highlight the principal steps. Setting Operating room at an academic medical center. Patients or Participants An asymptomatic morbidly obese (BMI 45) 37-year-old G6P1041 at 9 weeks and 5 days gestation by her LMP who was sent to the emergency room for further management after a transvaginal ultrasound confirmed her gestational age and detected a 4.8cm x 4.9cm x 4.8cm right-sided interstitial ectopic pregnancy with cardiac activity. She was hemodynamically stable with a beta-hCG of 47,458. She was considered a poor candidate for medical management and was taken to the operating room. Interventions Robotic-assisted laparoscopic resection of a cornual ectopic pregnancy using vasopressin, the purse-string circumferential suture, and resection of the ipsilateral fallopian tube with securement of collateral blood vessels as hemostatic techniques. Measurements and Main Results The robotic resection took approximately 98 minutes to complete with no complications and minimal blood loss. The patient met all postoperative milestones and was discharged from the hospital on postoperative day 1 in stable condition. Her follow up beta-hCG on postoperative day 3 was 3,034. Interestingly, the final pathology resulted in myometrium with a placenta increta with negative margins. Conclusion Minimally invasive techniques for the management of cornual ectopic pregnancies have continued to advance as more institutions are adopting these methods. Our case demonstrates that robotic-assisted resections of a cornual pregnancy can achieve minimal blood loss, adequate multi-layer myometrial closure, reduced postoperative complications, and overall safety. Our case highlights the necessity for a complete and careful resection of the myometrium given the very small, but possible association of a placenta accreta spectrum disorder even at this early gestational age. To demonstrate the robotic-assisted laparoscopic surgical technique in the resection of an advanced cornual ectopic pregnancy in a morbidly obese patient. Video recording of the cornual ectopic resection edited to highlight the principal steps. Operating room at an academic medical center. An asymptomatic morbidly obese (BMI 45) 37-year-old G6P1041 at 9 weeks and 5 days gestation by her LMP who was sent to the emergency room for further management after a transvaginal ultrasound confirmed her gestational age and detected a 4.8cm x 4.9cm x 4.8cm right-sided interstitial ectopic pregnancy with cardiac activity. She was hemodynamically stable with a beta-hCG of 47,458. She was considered a poor candidate for medical management and was taken to the operating room. Robotic-assisted laparoscopic resection of a cornual ectopic pregnancy using vasopressin, the purse-string circumferential suture, and resection of the ipsilateral fallopian tube with securement of collateral blood vessels as hemostatic techniques. The robotic resection took approximately 98 minutes to complete with no complications and minimal blood loss. The patient met all postoperative milestones and was discharged from the hospital on postoperative day 1 in stable condition. Her follow up beta-hCG on postoperative day 3 was 3,034. Interestingly, the final pathology resulted in myometrium with a placenta increta with negative margins. Minimally invasive techniques for the management of cornual ectopic pregnancies have continued to advance as more institutions are adopting these methods. Our case demonstrates that robotic-assisted resections of a cornual pregnancy can achieve minimal blood loss, adequate multi-layer myometrial closure, reduced postoperative complications, and overall safety. Our case highlights the necessity for a complete and careful resection of the myometrium given the very small, but possible association of a placenta accreta spectrum disorder even at this early gestational age.

Abstract Hormone receptor positive (HR+) breast cancers that express estrogen receptor (ER) can recur 5-20 years after initial diagnosis and treatment, often as bone metastases. More than 30% of patients with early-stage HR+ breast cancer treated with endocrine or hormonal therapy will relapse and all patients with metastatic breast cancer expressing ER eventually acquire resistance to endocrine therapy. The metastases are caused by slowly dividing well-differentiated cancer cells. These cells are not susceptible to chemotherapy, which only acts on dividing cells, and they are also resistant to endocrine therapy. Recent analyses indeed show that chemotherapy added to endocrine therapy for patients with lower risk early stage breast cancer did not increase overall survival. Thus, patients with metastatic HR+ breast cancer need new therapeutic options. Unfortunately HR+ breast cancers have not responded to current T-cell based immunotherapies. Therefore the objective of this study is to target HR+ breast cancers using a natural killer (NK) cell based approach. The type I IGF receptor (IGF1R) is expressed on hormone receptor positive (HR+) breast cancers and its expression is tightly related to ER function. We hypothesized that a drug that would target NK cells to IGF1R expressing HR+ cells will be effective in eliminating them. We generated a novel NK cell based immunotherapy called Tri specific Killer Engagers (TriKE) and these are much smaller molecules than conventional trispecific antibodies. The IGF1R TriKE consists of humanized single domain antibody (VHH) sequence of a camelid nanobody against CD16 to engage and activate NK cells, humanized VHH sequence of a nanobody against IGF1R to target HR+ breast cancer cells, and an IL-15 molecule between the two single domain antibodies to drive NK cell priming, expansion and survival. The IGF1R TriKE specifically bound to breast cancer cells expressing IGF1R and did not bind mouse embryonal fibroblasts from IGF1R knock out mice (R- cells). It bound R-/IGF1R cells (R- cells engineered to express human IGF1R) indicating specific binding to IGF1R on cells. The IGF1R TriKE did not cause downregulation of cell surface IGF1R levels in multiple HR+ breast cancer cells. The IGF1R TriKE specifically induced NK cell degranulation, measured by surface CD107a expression, against IGF1R expressing HR+ target breast cancer cells (MCF-7, T-47D and ZR-75-1) in a functional assay compared to treatment with anti-IGF1R nanobody or IL-15 alone. It did not induce degranulation of NK cells against R- cells but enhanced degranulation against R-/IGF1R cells suggesting that the functionality was specific for IGF1R expressing targets. Furthermore, the IGF1R TriKE also enhanced cell killing of ZR-75-1 breast cancer cells compared to IGF1R nanobody as measured by number of live breast cancer cells at the end of a 48 hour cytotoxicity assay. We are currently testing the IGF1R TriKE’s functionality in causing degranulation of NK cells from blood of breast cancer patients. These data indicate that this NK cell based immunotherapeutic strategy could be an effective treatment for HR+ metastatic breast cancer and make them responsive to immunotherapy. Our approach generates targeted off-the-shelf immunotherapy that does not require a personalized approach for each patient that is required with CAR T-cell based approaches that is expensive and time consuming. Citation Format: Emily Chiu, Ivy R Johnson, Jeffrey S Miller, Martin Felices, Deepali Sachdev. Targeting hormone receptor positive breast cancer for immune destruction by natural killer cells [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 P5-04-11.

In recent years, there have been significant advances in the treatment of breast cancer resulting in remarkably high survival rates. However, treatment options for metastatic triple negative breast cancer (TNBC) are quite limited due to a lack of identifiable, unique markers. Using a phage display-based whole cell biopanning procedure, we developed two human antibodies that bind to tumor cells with a metastatic TNBC phenotype. Our studies further identified domain 1 of HSPG2 (perlecan) protein as the cognate cell surface antigen bound by the antibody. Immunohistochemistry studies utilizing patient tissue samples revealed significant cell surface expression of HSPG2 in both primary tumors and metastatic lesions. Further, higher HSPG2 expression correlated with poor survival in TNBC. The affinity-matured antibody inhibited the growth of triple negative MDA-MB-231 tumors to a greater extent in nude mice than in NSG mice, pointing to the potential role of natural killer cell-mediated antibody-dependent cell cytotoxicity. This mechanism of action was confirmed through in vitro assays using mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). These results suggest that HSPG2 is a promising target in metastatic TNBC and HSPG2-targeted antibodies could represent a potentially novel class of targeted therapeutics for TNBC.

Abstract Estrogen receptor positive (ER+) breast cancers are treated with hormonal therapies such as tamoxifen or aromatase inhibitors. More than 30% of patients with early-stage ER+ breast cancer treated with endocrine therapy will relapse and all patients with metastatic breast cancer expressing ER eventually acquire resistance to hormonal therapy. Endocrine therapy resistant patients therefore require novel therapeutic options. Insulin-like growth factors (IGFs) and insulin signaling via the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively regulate breast cancer biology. Unfortunately, IGF1R targeted therapies failed to show a benefit in prolonging either disease-free or overall survival in clinical trials. In pre-clinical models acquired resistance to tamoxifen results in loss of IGF1R and enhanced sensitivity to IR signaling. Inhibition of mTOR alone relieves the negative feedback loop regulating levels of the adaptor protein IRS-1, which mediates proliferative effects of IGFs and insulin and enhanced phosphorylation of Akt. The ribosomal protein S6 kinase (S6K) phosphorylates IRS-1 on serine residues targeting it for proteasomal degradation, and this negative feedback regulation is important in attenuating IGF and insulin signaling. Cyclin dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. CDK4/6 inhibitors have recently been approved for treatment of ER+, Her2- advanced breast cancers and these such as palbociclib and abemaciclib block phosphorylation of retinoblastoma. IGFs and insulin stimulate cell cycle progression and increase cyclin D1 levels in breast cancers. Therefore, we hypothesized that CDK4/6 inhibition combined with IGF1R or IR targeting, to block mitogenic functions of IGF or insulin signaling, could be a viable therapeutic option in endocrine sensitive and endocrine resistant breast cancer, respectively. Parental MCF-7 and T47D were more sensitive to palbociclib compared to matched cells with acquired resistance to tamoxifen, MCF-7/TamR and T47D/TamR. Palbociclib also blocked IGF-I and insulin stimulated entry into cell cycle leading to G0/G1 arrest in ER+ breast cancer cells. Unlike mTOR inhibitors that upregulated IRS-1 levels leading to increased phosphorylation of Akt through IGF1R/IR, palbociclib did not affect IRS-1 levels and did not enhance phosphorylation of Akt in ER+ breast cancer cells. Combination of palbociclib with an IGF1R inhibitory antibody (huEM164), but not IR antibody (83-7), was better at inhibiting growth of endocrine sensitive MCF-7 and T-47D cells than either drug alone. Further, in a formal synergy study using the method of Chou-Talalay, the combination index of palbociclib and the IGF1R antibody was <1 for MCF-7 parent cells, indicating the two drugs synergistically inhibit growth. Combination of palbociclib with an IR antibody synergistically inhibited the growth of endocrine resistant MCF-7/TamR cells. Our data show that cotrageting CDK4/6 and IGF1R is more effective in endocrine sensitive but cotargeting CDK4/6 and IR is more effective in tamoxifen resistant breast cancer cells. These data indicate that targeting CDK4/6 and IR could be a therapeutic option for patients with endocrine resistant disease. Citation Format: Hoff K, Sachdev D. Targeting CDK4/6 and IGF1R or insulin receptor synergistically inhibits growth of endocrine sensitive and endocrine resistant breast cancers [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 P5-04-20.

Abstract Luminal breast cancers account for ~75% of all cases and while adjuvant hormone therapy targeting ERα has significantly improved overall survival for patients with ER+ tumors, acquired resistance remains a major clinical problem. Tamoxifen-resistant (TamR) breast cancer models show loss of IGF1R concomitant with increased insulin-induced growth, underpinned by insulin receptor (InsR) compensation for IGF1R loss. PR (gene name PGR) is an estrogen-regulated target gene whose expression is used as a clinical marker of ER activity. However, its de novo relevance to breast cancer is unclear. We have shown previously that post-translational modifications create unique PR species whose altered behavior drives an endocrine-resistant gene signature, in part by crosstalk with the IGFR pathway. We propose that phospho-PR target gene selectivity is mediated by cooperation between PR-B and InsR/IGF1R pathway components. Herein we show that phospho-PR-expressing T47D cells lose expression of IGF1R compared to control cells expressing WT PR-B. Furthermore, expression of the adapter protein IRS-1 requires PRB expression and these IRS-1+ cells were more sensitive to insulin in anchorage-dependent growth assays. Phospho-PR T47D cells exhibit increased ALDH+ and CD24-/CD44+ tumorsphere formation compared to wt PRB-expressing cells. Inhibitors targeting IRS-1 and InsR were used to test their requirement for tumorsphere growth and, surprisingly, IRS-1 perturbation reduced phospho-PRB but not wt PRB tumorsphere growth. Interestingly, IRS-1 replaces IGF1R in phospho-PRB-containing transcriptional complexes that are recruited to the CTSD promoter, which we previously identified as a phospho-PR target gene. Finally, breast cancer cells expressing phospho-PR species exhibited tamoxifen-resistant growth. Collectively our data suggest that phospho-PRB cooperates with IRS-1 downstream of the Ins/IGF1R system to promote outgrowth of endocrine-resistant cells that include ALDH+ stem-like cells capable of forming secondary tumorspheres in vitro. Targeting phospho-PR species in addition to the signaling components of PR-complexes may provide a means to block emergence of endocrine-resistant cancer cells during breast cancer progression. Citation Format: Amy R. Dwyer, Deepali Sachdev, Carol A. Lange. Progesterone receptor/IRS-1 cooperation promotes stem cell outgrowth and endocrine resistance in estrogen receptor-positive luminal breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 949.

Abstract Insulin-like growth factors (IGFs) and insulin signaling via the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively, are potent activators of PI3K/Akt/mTOR. Drugs targeting IGF1R and the related IR were tested clinically including in combination with mTOR inhibitors. Inhibition of mTOR was not effective as inhibition of mTOR relieved the negative feedback loop regulating levels of the adaptor protein, insulin receptor substrate 1 (IRS-1), that mediates proliferative effects of IGFs and insulin and rapamycin enhanced phosphorylation of Akt. The ribosomal protein S6 kinase (S6K) phosphorylates IRS-1 on serine residues and targets it for proteasomal degradation, and this negative feedback regulation is important in attenuating IGF and insulin signaling. Cyclin dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. CDK4/6 inhibitors have recently been approved for treatment of estrogen receptor positive (ER+), Her2- advanced breast cancers and these CDK4/6 inhibitors such as palbociclib block phosphorylation of retinoblastoma (Rb). IGFs and insulin stimulate cell cycle progression and increase cyclin D1 levels in breast cancers. Therefore, we hypothesized that CDK4/6 inhibition can be combined with IGF1R/IR targeting to block mitogenic functions of IGF/insulin signaling in breast cancer as this would not relieve the negative feedback regulation of IGF and insulin signaling. Palbociclib blocked growth of ER+ parental MCF-7 and T47D breast cancer cells that respond to hormonal therapy including tamoxifen, a selective estrogen receptor modulator. Parental MCF-7 and T47D were more sensitive to palbociclib compared to matched cells with acquired resistance to tamoxifen (MCF-7/TamR and T47D/TamR). Palbociclib also blocked IGF-I and insulin stimulated entry into cell cycle leading to G0/G1 arrest in ER+ breast cancer cells. Combination of palbociclib with an IGF1R inhibitory antibody, but not IR antibody, was more effective in inhibiting growth of tamoxifen sensitive parental ER+ breast cancer cells. In contrast combination of palbociclib with an IR antibody was effective in inhibition of MCF-7/TamR cells. Unlike mTOR inhibitors that upregulated IRS-1 levels leading to increased phosphorylation of Akt through IGF1R/IR, palbociclib did not affect IRS-1 levels and did not enhance phosphorylation of Akt in ER+ and TNBC cells. Further, palbociclib also blocked growth and cell cycle progression of triple negative breast cancer (TNBC) cells. Our data show that combining IGF1R/IR inhibitors with palbociclib may be superior to combining them with mTOR inhibitors for ER+ breast cancer and palbociclib can be a potential therapeutic strategy for TNBC. These data implicate that cotargeting CDK4/6 and IR could be a therapeutic option for patients with endocrine resistant disease. Citation Format: Katelyn Hoff, Deepali Sachdev. CDK4/6 inhibition blocks effects of IGFs and insulin in estrogen receptor positive and triple negative breast cancers [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 2303.

IGF and insulin signaling via the type I insulin-like growth factor receptor (IGF1R) and insulin receptor (IR) respectively, are potent activators of PI3K/Akt/mTOR. Drugs targeting IGF1R and the related IR were tested clinically including in combination with mTOR inhibitors. Inhibition of mTOR was not effective as inhibition of mTOR relieved the negative feedback loop regulating levels of the adaptor protein, insulin receptor substrate 1 (IRS-1), that mediates proliferative effects of IGFs and insulin and rapamycin enhanced phosphorylation of Akt. The ribosomal protein S6 kinase (S6K) phosphorylates IRS-1 on serine residues and targets it for proteasomal degradation and this negative feedback regulation is important in attenuating IGF and insulin signaling. Cyclin dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. CDK4/6 inhibitors have recently been approved for treatment of estrogen receptor positive (ER+), Her2- advanced breast cancers and these CDK4/6 inhibitors such as palbociclib block phosphorylation of retinoblastoma (Rb). IGFs and insulin stimulate cell cycle progression and increase cyclin D1 levels in breast cancers. Therefore, we hypothesized that CDK4/6 inhibition can be combined with IGF1R/IR targeting to block mitogenic functions of IGF/insulin signaling in breast cancer as this would not relieve the negative feedback regulation of IGF and insulin signaling. Herein, we analyzed the effect of palbociclib on IGF-I/insulin signaling, Rb phosphorylation and growth of various subtypes of breast cancer cells. Palbociclib blocked growth of both endocrine sensitive and resistant ER+ breast cancers. Further, ER+ parental MCF-7 and T47D cells that respond to hormonal therapy including tamoxifen, a selective estrogen receptor modulator, were more sensitive to palbociclib compared to matched cells with acquired resistance to tamoxifen. Palbociclib blocked IGF-I and insulin stimulated entry into cell cycle leading to G0/G1 arrest in ER+ breast cancer cells. Further, palbociclib also blocked growth and cell cycle progression of triple negative breast cancer (TNBC) cells. Unlike mTOR inhibitors that upregulated IRS-1 levels leading to increased phosphorylation of Akt through IGF1R/IR, palbociclib did not affect IRS-1 levels and did not enhance phosphorylation of Akt in ER+ and TNBC cells. Further, combination of palbociclib with IGF1R inhibitory antibody was more effective in inhiibiting growth of ER+ breast cancer cells. Our data show that palbociclib can be a potential therapeutic strategy for TNBC and that combining IGF1R/IR inhibitors with palbociclib may be superior to combining them with mTOR inhibitors for ER+ breast cancer. Citation Format: Sachdev D, Hoff K. CDK4/6 inhibition blocks effects of IGFs and insulin in estrogen receptor positive and triple negative breast cancers: Implications for cotargeting IGF1R/IR and CDKs [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-03.

Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1). ALDH expression significantly promotes tumor initiation and correlates with the levels of EMP2 expression in both patient samples and tumor cell lines. As therapy against cancer stem cells in endometrial cancer is lacking, the ability of anti-EMP2 IgG1 therapy to reduce primary and secondary tumor formation using xenograft HEC1A models was determined. Anti-EMP2 IgG1 reduced the expression and activity of ALDH and correspondingly reduced both primary and secondary tumor load. Our results collectively suggest that anti-EMP2 therapy may be a novel method of reducing endometrial cancer stem cells.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been implicated in promoting breast cancer growth and progression. While the autocrine effects of FGFR activation in tumor cells have been extensively studied, little is known about the effects of tumor cell-derived FGFs on cells in the microenvironment. Because FGF signaling has been implicated in the regulation of bone formation and osteoclast differentiation, we hypothesized that tumor cell-derived FGFs are capable of modulating osteoclast function and contributing to growth of metastatic lesions in the bone. Initial studies examining FGFR expression during osteoclast differentiation revealed increased expression of FGFR1 in osteoclasts during differentiation. Therefore, studies were performed to determine whether tumor cell-derived FGFs are capable of promoting osteoclast differentiation and activity. Using both non-transformed and transformed cell lines, we demonstrate that breast cancer cells express a number of FGF ligands that are known to activate FGFR1. Furthermore our results demonstrate that inhibition of FGFR activity using the clinically relevant inhibitor BGJ398 leads to reduced osteoclast differentiation and activity in vitro. Treatment of mice injected with tumor cells into the femurs with BGJ398 leads to reduced osteoclast activity and bone destruction. Together, these studies demonstrate that tumor cell-derived FGFs enhance osteoclast function and contribute to the formation of metastatic lesions in breast cancer.

Type 2 diabetes (T2D) is associated with increased cancer risk and cancer-related mortality. Data herein show that we generated an immunodeficient hyperinsulinemic mouse by crossing the Rag1(-/-) mice, which have no mature B or T lymphocytes, with the MKR mouse model of T2D to generate the Rag1(-/-) (Rag/WT) and Rag1(-/-)/MKR(+/+) (Rag/MKR) mice. The female Rag/MKR mice are insulin resistant and have significantly higher nonfasting plasma insulin levels compared with the Rag/WT controls. Therefore, we used these Rag/MKR mice to investigate the role of endogenous hyperinsulinemia on human cancer progression. In this study, we show that hyperinsulinemia in the Rag/MKR mice increases the expression of mesenchymal transcription factors, TWIST1 and ZEB1, and increases the expression of the angiogenesis marker, vascular endothelial growth factor A (VEGFA). We also show that silencing the insulin receptor (IR) in the human LCC6 cancer cells leads to decreased tumor growth and metastases, suppression of mesenchymal markers vimentin, SLUG, TWIST1 and ZEB1, suppression of angiogenesis markers, VEGFA and VEGFD, and re-expression of the epithelial marker, E-cadherin. The data in this paper demonstrate that IR knockdown in primary tumors partially reverses the growth-promoting effects of hyperinsulinemia as well as highlighting the importance of the insulin receptor signaling pathway in cancer progression, and more specifically in epithelial-mesenchymal transition.

IGF1R and insulin receptor (IR) regulate biology of estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. The results of initial clinical trials with anti-IGF1R drugs have been disappointing due to feedback upregulation of insulin receptor (IR) signaling and absence of biomarkers for these drugs. We evaluated the effects of BMS-754807, a dual tyrosine kinase inhibitor of IGF1R/IR on ER+ and TNBC. In ER+ MCF-7 cells, it inhibited xenograft growth of MCF-7 tumors (n = 10 per treatment) compared to vehicle. Interestingly, while tumor growth was suppressed over a period of five weeks, eventually the tumors displayed resistance to BMS-754807. In TNBC, it inhibited motility in vitro. In contrast to ER+ cells, BMS-754807 did not inhibit primary tumor growth of TNBC cells injected into the mammary fat pad of mice. But at a dose of 50 mg/kg daily inhibited metastasis of TNBC cells, MDA-231-LM2 and MDA-MB435A/LCC6 cells, in the orthotopic and tail vein models of metastasis compared to vehicle (n = 10/group). Our data indicate that regulation of metastasis and tumor growth by IGF1R can be discrete events and functional imaging to identify biological properties of metastatic breast cancer regulated by IGF1R/IR are needed to better define treatments. While MRI is a powerful tool for detecting and imaging cancer, its utility in imaging metastasis to the lung is limited due to the challenges of lung MRI with conventional 3D gradient echo (GRE). MRI does not visualize lung well, mainly due to the abundance of air-tissue interfaces, which cause the MR signal to decay too rapidly for conventional MRI pulse sequences to capture. Clinically metastases are monitored by CT or PET but exposure of patients to ionizing radiation is a concern and problematic in longitudinal studies monitoring response to a targeted drug. Therefore, we recently reported the utility of a novel MR sequence called sweep imaging with Fourier transformation (SWIFT), where the data is acquired quasi-simultaneously with the radiofrequency pulse, to image lung metastasis of breast cancer. Here, we monitored response to IGF1R and IGF1R/IR targeted drugs in preclinical models of lung metastasis of breast cancer. We used MDA-231-LM2 cells with the tail vein injection model of metastasis. Mice with breast cancer metastases in the lungs were treated with either huEM164, an antibody against IGF1R, or BMS-754807. Metastasis was monitored by BLI and SWIFT MRI weekly. SWIFT was more sensitive in detecting inhibition of metastasis by these drugs. Thus, dual inhibition of IGF1R and IR is effective in blocking growth of ER+ and metastasis of TNBC. However, combination of this therapeutic strategy with other agents may be necessary to prevent or delay onset of resistance. Further, noninvasive biomarkers of response to IGF1R/IR targeted drugs can be developed with SWIFT imaging. Citation Format: Deepali Sachdev, Huy Donguyen, Naoharu Kobayashi, Sidath C. Kumarapperuma, Joeseph C. Weber. Functional imaging markers for blockade of breast cancer metastasis by IGF1R and insulin receptor targeted drugs using novel MRI and targeted iron oxide nanoparticles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4248.

Insulin-like growth factors (IGFs) and insulin acting via the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively regulate biology of estrogen positive (ER+) and triple negative (TN) breast cancer cells. In animal models, inhibition of IGF1R alone with antibodies has demonstrated significant inhibition of tumor growth and/or metastasis of several types of cancer cells. Unfortunately, the results of initial clinical trials with anti-IGF1R antibodies have been disappointing. One reason for this is that inhibition of the related IR through which insulin and IGF-II signal, may be necessary for optimal efficacy of this targeted approach. Second, there is a need to develop markers that can be used to stratify patients who may benefit from these drugs and/or monitor response to these drugs. To examine the effectiveness of dual inhibition of IGF1R and IR, we evaluated the effects of BMS-754807, a small molecule dual tyrosine kinase inhibitor of IGF1R/IR on ER+ and TN breast cancer cells. In ER+ cells (MCF-7, T47D and ZR-75-1), BMS-754807 inhibited IGF-I, IGF-II and insulin stimulated activation of downstream PI3K and MAPK pathways, proliferation and anchorage-independent growth in vitro. BMS-754807 also blocked signaling in MCF-7 tumors and inhibited xenograft growth of MCF-7 tumors (n=10 per treatment) compared to vehicle. Interestingly, while tumor growth was suppressed over a period of five weeks, eventually the tumors displayed resistance to BMS-754807. In TN cell lines (MDA-MB-231; MDA-MB-231-LM2 and MDA-231-BoM, lung seeking and bone specific metastatic variants respectively of MDA-MB-231; and MDA-MB435A/LCC6) BMS-754807 also inhibited activation of the PI3K pathway and motility in vitro. In contrast to ER+ cells, BMS-754807 did not inhibit primary tumor growth of TN breast cancers cells injected into the mammary fat pad of mice. But BMS-754807 (50 mg/kg daily by oral gavage) inhibited metastasis of TN cells, MDA-231-LM2 and MDA-MB435A/LCC6 cells, in the orthotopic and tail vein models of metastasis compared to vehicle (n=10/group). Our data indicate that regulation of metastases and tumor growth by IGF1R can be discrete events and functional imaging to identify biological properties of metastatic breast cancer regulated by IGF1R/IR are needed to better define treatments. Therefore, we used a novel MR sequence called sweep imaging with Fourier transformation (SWIFT), where the data is acquired quasi-simultaneously with the radiofrequency pulse, to monitor response to BMS-754807. We used MDA-231-LM2 cells with the tail vein injection model of metastasis. Mice injected with breast cancer cells were treated with either vehicle or BMS-754807 and metastasis was monitored by BLI and SWIFT MRI weekly. SWIFT was sensitive in detecting inhibition of metastasis by BMS-754807. Our results suggest that dual inhibition of IGF1R and IR is effective in blocking growth of ER+ and metastasis of TN breast cancers. However, combination of this therapeutic strategy with other agents may be necessary to prevent or delay onset of resistance. Further, noninvasive biomarkers of response to IGF1R/IR targeted drugs can be developed with SWIFT imaging. Citation Format: Deepali Sachdev, Joel D Winer, Naoharu Kobayashi, Michael Garwood, Joseph Weber. Dual inhibition of IGF1R and insulin receptor in estrogen receptor positive and triple negative breast cancer and monitoring blockade of metastasis using novel MRI [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-03.