Targeting IGF-1R Research Articles

Glioma Therapy: Oncogenic State and Cell Identity Combinatorially Dictate the Susceptibility of Cells within Glioma Development Hierarchy to IGF1R Targeting (Adv. Sci. 21/2020)

In article number 2001724, Chong Liu and co-workers describe that the oncogenic state and cell identity need to match precisely to sensitize a glioma cell to IGF1R targeting. This phenomenon parallels the Chinese mortise and tenon joint structure, where different wood pieces are connected precisely by their mortise and tenon joints. Destruction of the mortise-and-tenon work (resembles the glioma hierarchy) needs the knowledge of how such structure is built up.

Oncogenic State and Cell Identity Combinatorially Dictate the Susceptibility of Cells within Glioma Development Hierarchy to IGF1R Targeting.

Glioblastoma is the most malignant cancer in the brain and currently incurable. It is urgent to identify effective targets for this lethal disease. Inhibition of such targets should suppress the growth of cancer cells and, ideally also precancerous cells for early prevention, but minimally affect their normal counterparts. Using genetic mouse models with neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) as the cells‐of‐origin/mutation, it is shown that the susceptibility of cells within the development hierarchy of glioma to the knockout of insulin‐like growth factor I receptor (IGF1R) is determined not only by their oncogenic states, but also by their cell identities/states. Knockout of IGF1R selectively disrupts the growth of mutant and transformed, but not normal OPCs, or NSCs. The desirable outcome of IGF1R knockout on cell growth requires the mutant cells to commit to the OPC identity regardless of its development hierarchical status. At the molecular level, oncogenic mutations reprogram the cellular network of OPCs and force them to depend more on IGF1R for their growth. A new‐generation brain‐penetrable, orally available IGF1R inhibitor harnessing tumor OPCs in the brain is also developed. The findings reveal the cellular window of IGF1R targeting and establish IGF1R as an effective target for the prevention and treatment of glioblastoma.

49P Targeting IGF1R in osteosarcoma

49P Targeting IGF1R in osteosarcoma

Abstract CT195: A phase 2 clinical trial of palbociclib and ganitumab for relapsed/refractory Ewing sarcoma

Abstract Background: Ewing sarcoma is a classic translocation-associated malignancy, with nearly all cases carrying an identifiable EWSR1 or FUS translocation. The translocation leads to an aberrant fusion oncoprotein that is thought to function as a pathogenic transcription factor. Despite our understanding of the fundamental biology of this disease, targeting this transcription factor has been elusive. The insulin-like growth factor receptor (IGF-1R) pathway has been long implicated in the pathogenesis of Ewing sarcoma based on early findings that IGF-1 and IGF-1R are overexpressed in Ewing sarcoma. A range of preclinical studies have demonstrated the activity of IGF-1R targeting antibodies in multiple Ewing sarcoma cell lines. Multiple early phase studies of IGF-1R inhibitors have demonstrated that approximately 10% of patients with relapsed disease respond to these agents as monotherapy. Recent work has identified CDK4 as a genomic vulnerability in Ewing sarcoma. Given that CDK4/6 inhibitor monotherapy is prone to resistance, a series of investigations were undertaken to identify potential combination approaches. In an open reading frame experiment designed to determine whether over expression of specific genes would confer resistance, IGF-1R scored highly. Subsequent in vitro and in vivo experiments demonstrated synergy between CDK4/6 inhibition and IGF-1R inhibition. We have translated these findings into an open phase 2 clinical trial of ganitumab, a monoclonal antibody inhibitor of IGF-1R, and palbociclib, a small molecule inhibitor of CDK4/6. Methods: This is a Phase 2, single-arm, single-stage, investigator-initiated clinical trial of palbociclib and ganitumab in patients 12-50 years of age with relapsed/refractory Ewing sarcoma (NCT04129151). The primary objectives are to estimate the objective radiographic response rate to the combination of palbociclib and ganitumab and to describe the toxicity of this drug combination in patients with relapsed/refractory Ewing sarcoma. Palbociclib is given at a dose of 100 mg PO on days 1-21 of a 28-day cycle. Ganitumab is given via IV at a dose of 18 mg/kg on days 1 and 15. The primary endpoint is objective response by RECIST, with disease evaluated after every 2 cycles. Up to 18 patients will be enrolled to yield 15 response evaluable patients. With 15 evaluable patients the study will have 91% power and a type-1 error rate of 0.056 to determine whether the response rate in this population is greater than 40% and significantly different from the 10% response rate seen with IGF-1R monotherapy. Pharmacodynamic testing for confirmation of IGF-1R inhibition (serum IGF-related proteins) is required for all patients. Circulating tumor DNA (ctDNA) is measured at study entry, on cycle 1 day 15, at start of cycle 2 and at each disease evaluation. ctDNA levels will be measured using next generation sequencing for quantification of ctDNA and association with response, and for identification of genomic and epigenetic markers of resistance. Enrollment began in December 2019. Citation Format: David S. Shulman, Katherine Thornton, Edwin Choy, Lillian M. Guenther, Kerri Cavanaugh, Megan Forsyth, Kylene DeSmith, Catherine Clinton, Kimberly Stegmaier, Brian Crompton, Wendy B. London, Steven G. DuBois. A phase 2 clinical trial of palbociclib and ganitumab for relapsed/refractory Ewing sarcoma [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 CT195.

IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. The insulin-like growth factor (IGF) system plays a key role in regulating growth and invasiveness in several malignancies, including ovarian cancer. IGF1R targeting showed antiproliferative activity of EOC cells. However, clinical studies failed to show significant benefit. EOC cells suppress antitumor immune responses by inducing dendritic cell (DC) dysfunction. The IGF1 axis can regulate DC maturation.The current study evaluated involvement of the IGF1 axis in DC differentiation in EOC. Studies were conducted on EOC and on a human monocyte cell line. Tissue microarray analysis (TMA) was performed on 36 paraffin blocks from EOC patients. Expression of IGF1R, p53, Ki67, BRCA1, and DC markers was evaluated using immunohistochemistry. Co-culture of EOC cells with DC pretreated with IGF1R inhibitor blocked cancer cell migration. TMA demonstrated higher rate of IGF1R protein expression in patients with advanced (76.9%) as compared to early (40%) EOC. A negative correlation between IGF1R protein expression and the CD1c marker was found. These findings provide evidence that IGF1R axis inhibition could be a therapeutic strategy for ovarian cancer by restoring DC-mediated antitumor immunity.

MicroRNA‐379 mediates pigmentation, migration and proliferation of melanocytes by targeting the insulin‐like growth factor 1 receptor

Melanogenesis, migration and proliferation of melanocytes are important factors that determine the hair colours of mammals. MicroRNAs (miRNAs) have been shown to be closely related to these processes. In melanocytes of alpacas, insulin-like growth factor 1 (IGF1) has been shown to improve melanogenesis through the cyclic AMP (cAMP) pathway. miR-379 was predicted to target insulin-like growth factor (IGF) receptor 1 (IGF1R), which binds to IGF1. Therefore, we hypothesized that miR-379 could mediate melanogenesis, migration and proliferation of melanocytes. Here, we report that miR-379 was highly expressed in alpaca melanocytes. Subsequent overexpression of miR-379 in alpaca melanocytes led to the generation of the phenotype of melanogenesis, proliferation and migration. In addition, the expression of genes related to these phenotypes in melanocytes was detected. Our results showed that miR-379 targets IGF1R in melanocytes. The overexpression of miR-379 stimulated dendrite extension or elongation and limited the perinuclear distribution of melanin, but inhibited melanogenesis via cAMP response element (CRE)-binding protein (CREB)/microphthalmia-associated transcription factor (MITF) pathway. miR-379 attenuated melanocyte migration by downregulating the focal adhesion kinase (FAK) and enhanced melanocyte proliferation by upregulating protein kinase B (AKT). These observations suggest the involvement of miR-379 in the physiological regulation of melanocytes, mediated by targeting IGF1R on insulin receptor (IR) compensation and subsequent crosstalk.

MiR-99b regulates cerebral ischemia neuronal injury through targeting IGF1R.

Recently, microRNA-99b (miR-99b) shows diverse functions in different human disease. However, further studies about the potential effect of miR-99b in cerebral ischemia injury still need to be done. The expressions of miR-99b and IGF1R were detected via RT-qPCR assay. Western blot assay was applied to measure the protein expression of Caspase-3, Bax and Bcl-2. MTT assay was used to observe cell viability of SH-SY5Y cells. The association of miR-99b and IGF1R was testified by dual luciferase assay. And human SH-SY5Y cells were treated with the oxygen-glucose deprivation/reperfusion (OGD/R) to mimic CIR injury. The expression of miR-99b was increased in the OGD/R model. And upregulation of miR-99b promoted cell viability and inhibited apoptosis induced by OGD/R. Moreover, IGF1R was confirmed as a direct target gene of miR-99b. The expression of IGF1R was obviously decreased under OGD/R conditions. MiR-99b promoted the viability and suppressed apoptosis of SH-SY5Y cells under OGD/R conditions through targeting IGF1R.

Dual targeting of IGF-1R and ErbB3 as a potential therapeutic regimen for ovarian cancer

Therapeutically targeting receptor tyrosine kinases has proven to be paramount to overcoming chemotherapy resistance in several cancer indications, improving patient outcomes. Insulin-Like Growth Factor Receptor 1 (IGF-1R) and Epidermal Growth Factor Receptor 3 (ErbB3) have been implicated as two such drivers of resistance, however their simultaneous role in ovarian cancer chemotherapy resistance remains poorly elucidated. The aim of this work is to determine the effects of dual IGF-1R/ErbB3 inhibition on ovarian cancer cell signaling, growth, and in vivo efficacy. Assessment of in vitro chemotherapy response across a panel of ovarian cancer cell lines revealed that increased IGF-1R cell surface expression correlates with decreased sensitivity to chemotherapy, and that growth induced by IGF-1R and ErbB3 ligands is blocked by the tetravalent bispecific antibody targeting IGF-1R and ErbB3, istiratumab. In vitro chemotherapy treatment increased ovarian cancer cell line capacity to activate prosurvival PI3K signaling in response to ligand, which could be prevented with istiratumab treatment. Furthermore, in vivo efficacy of standard of care chemotherapies using a xenograft model of ovarian cancer was potentiated with istiratumab. Our results suggest a role for IGF-1R and ErbB3 in driving chemotherapy resistance of ovarian cancer.

1942P - Preclinical evaluation targeting both IGF1R and IR in triple negative breast cancer

BackgroundInsulin Receptor (INSR) signalling may play a role in resistance to insulin-like growth factor receptor (IGF1R) therapy. Although IGF1R is frequently expressed in triple negative breast cancer (TNBC), we found that an IGF1R monoclonal antibody did not inhibit the growth of TNBC cells. Thus, we hypothesised that dual targeting of IGF1R and INSR may be more effective in TNBC. MethodsRelative mRNA expression of INSR and IGFIR were analysed across different subtypes of breast cancer using the BreastMark database. INSR-A, INSR-B, IGF1R and insulin like growth factor 2 (IGFII) was quantified by qRT-PCR in a panel of 11 TNBC cell lines. Proliferation assays were performed on TNBC cell lines treated with the dual IR/INSR inhibitor Linsitinib (OSI-906). Combinations were performed by testing Linsitinib with Xentuzumab (BI-836845), cisplatin, docetaxel, or the PI3K inhibitor Dactolisib (NVP-BEZ235). We also tested the anti-proliferative effect of Linsitinib in combination with Xentuzumab when stimulated with IGF-I and IGF-II following serum-starvation for 24hours. Finally, Linsitinib was tested in HCC-1143 xenografts to assess the effect of low-dose treatment on tumour formation. ResultsNo significant differences were observed between the basal-like and non-basal-like cell lines for IR or IGF1R mRNA expression. However, IGF-II mRNA was more frequently detected in non-basal-like (80%, 4/5) compared to basal-like cell lines (20%, 1/5). Only three of the 12 cell lines tested showed sensitivity to Linsitinib with IC50 values less than 10 µM. The two most sensitive cell lines, HDQ-P1 and HCC1143, expressed detectable levels of IGF1R, INSR-A and INSR-B mRNAs. Although Linsitinib blocked IGF-I and IGF-II stimulated proliferation in both HCC1143 and HDQ-P1 cells, addition of Linsitinib to either chemotherapy or Dactolisib did not enhance therapeutic response. Linisitinib did not reduce tumour formation or tumour growth in an in vivo TNBC cell-line xenograft model. ConclusionsAlthough IGF1R has been shown to be frequently expressed in TNBC, our in vitro and in vivo data suggest that it may not be a good therapeutic target in the TNBC subtype. Legal entity responsible for the studyThe authors. FundingBoehringer Ingleheim. DisclosureN. O’Donovan: Research grant / Funding (institution): Boehringer Ingleheim. J. Crown: Honoraria (self), Speaker Bureau / Expert testimony, Research grant / Funding (institution): Boehringer Ingleheim; Shareholder / Stockholder / Stock options, Full / Part-time employment: OncoMark Limited; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Eisai; Honoraria (self): Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Puma Biotechnology; Honoraria (self), Advisory / Consultancy: Seattle Genetics; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy: Vertex; Honoraria (self), Speaker Bureau / Expert testimony: Genomic Health; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self), Travel / Accommodation / Expenses: MSD Oncology; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Abbvie. All other authors have declared no conflicts of interest.

Development of combination therapies to maximize the impact of KRAS-G12C inhibitors in lung cancer.

KRAS represents an excellent therapeutic target in lung cancer, the most commonly mutated form of which can now be blocked using KRAS-G12C mutant-specific inhibitory trial drugs. Lung adenocarcinoma cells harboring KRAS mutations have been shown previously to be selectively sensitive to inhibition of mitogen-activated protein kinase kinase (MEK) and insulin-like growth factor 1 receptor (IGF1R) signaling. Here, we show that this effect is markedly enhanced by simultaneous inhibition of mammalian target of rapamycin (mTOR) while maintaining selectivity for the KRAS-mutant genotype. Combined mTOR, IGF1R, and MEK inhibition inhibits the principal signaling pathways required for the survival of KRAS-mutant cells and produces marked tumor regression in three different KRAS-driven lung cancer mouse models. Replacing the MEK inhibitor with the mutant-specific KRAS-G12C inhibitor ARS-1620 in these combinations is associated with greater efficacy, specificity, and tolerability. Adding mTOR and IGF1R inhibitors to ARS-1620 greatly improves its effectiveness on KRAS-G12C mutant lung cancer cells in vitro and in mouse models. This provides a rationale for the design of combination treatments to enhance the impact of the KRAS-G12C inhibitors, which are now entering clinical trials.

MicroRNA-139 is a predictor of prostate cancer recurrence and inhibits growth and migration of prostate cancer cells through cell cycle arrest and targeting IGF1R and AXL.

We previously identified a panel of five microRNAs (miRNAs) associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using next-generation sequencing-based whole miRNome sequencing and quantitative polymerase chain reaction-based validation analysis. In this study, we examined the mechanism of action of miR-139-5p, one of the downregulated miRNAs identified in the panel. Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan-Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that overexpressed miR-139 to confirm its targets as well as examine pathways through which miR-139 may function using cell-based assays. Low miR-139 expression was significantly associated with a variety of prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity, and lymph node status. MiR-139 expression was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis were significantly lower among patients with high miR-139 expression (P = .0004 and .038, respectively). Validation in the TCGA data set showed a significant association between dichotomized miR-139 expression and biochemical recurrence (odds ratio, 0.52; 95% confidence interval, 0.33-0.82). Overexpression of miR-139 in prostate cancer cells led to a significant reduction in cell proliferation and migration compared with control cells, with cells arrested in G2 of cell cycle. IGF1R and AXL were identified as potential targets of miR-139 based on multiple miRNA-binding sites in 3'-untranslated regions of both the genes and their association with prostate cancer growth pathways. Luciferase assays verified AXL and IGF1R as direct targets of miR-139. Furthermore, immunoblotting of prostate cancer cells demonstrated IGF1R and AXL protein expression were inhibited by miR-139 treatment, which was reversed by the addition of miR-139 antagomir. Examination of the molecular mechanism of growth inhibition by miR-139 revealed the downregulation of activated AKT and cyclin D1, with upregulation of the CDK inhibitor p21. miR-139 is associated with improved prognosis in patients with localized prostate cancer, which may be mediated through downregulation of IGF1R and/or AXL and associated signaling pathway components.

GSTZ1‐1 Deficiency Activates NRF2/IGF1R Axis in HCC via Accumulation of Oncometabolite Succinylacetone

The IGF1R signaling is important in the malignant progression of cancer. However, overexpression of IGF1R has not been properly assessed in HCC. Here, we revealed that GSTZ1‐1, the enzyme in phenylalanine/tyrosine catabolism, is downregulated in HCC, and its expression was negatively correlated with IGF1R. Mechanistically, GSTZ1‐1 deficiency led to succinylacetone accumulation, alkylation modification of KEAP1, and NRF2 activation, thus promoting IGF1R transcription by recruiting SP1 to its promoter. Moreover, inhibition of IGF1R or NRF2 significantly inhibited tumor‐promoting effects of GSTZ1 knockout in vivo. These findings establish succinylacetone as an oncometabolite, and GSTZ1‐1 as an important tumor suppressor by inhibiting NRF2/IGF1R axis in HCC. Targeting NRF2 or IGF1R may be a promising treatment approach for this subset HCC.

Dual Targeting of EGFR and IGF1R in the TNFAIP8 Knockdown Non-Small Cell Lung Cancer Cells.

Aberrant regulation of EGFR is common in non-small cell lung carcinomas (NSCLC), and tumor resistance to targeted therapies has been attributed to emergence of other co-occurring oncogenic events, parallel bypass receptor tyrosine kinase pathways including IGF1R, and TNFα-driven adaptive response via NF-κB. TNFAIP8, TNFα-inducible protein 8, is an NF-κB-activated prosurvival and oncogenic molecule. TNFAIP8 expression protects NF-κB-null cells from TNFα-induced cell death by inhibiting caspase-8 activity. Here, we demonstrate that knockdown of TNFAIP8 inhibited EGF and IGF-1-stimulated migration in NSCLC cells. TNFAIP8 knockdown cells showed decreased level of EGFR and increased expression of sorting nexin 1 (SNX1), a key regulator of the EGFR trafficking through the endosomal compartments, and treatment with SNX1 siRNA partially restored EGFR expression in these cells. TNFAIP8 knockdown cells also exhibited downregulation of IGF-1-induced pIGF1R and pAKT, and increased expression of IGF-1-binding protein 3 (IGFBP3), a negative regulator of the IGF-1/IGF1R signaling. Consistently, treatment of TNFAIP8 knockdown cells with IGFBP3 siRNA restored pIGF1R and pAKT levels. TNFAIP8 knockdown cells had enhanced sensitivities to inhibitors of EGFR, PI3K, and AKT. Furthermore, IHC expression of TNFAIP8 was associated with poor prognosis in NSCLC. These findings demonstrate TNFAIP8-mediated regulation of EGFR and IGF1R via SNX1 and IGFBP3, respectively. We posit that TNFAIP8 is a viable, multipronged target downstream of the TNFα/NF-κB axis, and silencing TNFAIP8 may overcome adaptive response in NSCLC. IMPLICATIONS: TNFAIP8 and its effectors SNX1 and IGFBP3 may be exploited to improve the efficacy of molecular-targeted therapies in NSCLC and other cancers.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/5/1207/F1.large.jpg.

MiR-644a Disrupts Oncogenic Transformation and Warburg Effect by Direct Modulation of Multiple Genes of Tumor-Promoting Pathways

Castration-resistant prostate cancer (CRPC) is defined by tumor microenvironment heterogeneity affecting intrinsic cellular mechanisms including dysregulated androgen signaling, aerobic glycolysis (Warburg effect), and aberrant activation of transcription factors including androgen receptor (AR) and c-Myc. Using in vitro, in vivo, and animal models, we find a direct correlation between miR-644a downregulation and dysregulation of essential cellular processes. MiR-644a downregulated expression of diverse tumor microenvironment drivers including c-Myc, AR coregulators, and antiapoptosis factors Bcl-xl and Bcl2. Moreover, miR-644a modulates epithelial-mesenchymal transition (EMT) by directly targeting EMT-promoting factors ZEB1, cdk6, and Snail. Finally, miR-644a expression suppresses the Warburg effect by direct targeting of c-Myc, Akt, IGF1R, and GAPDH expression. RNA sequencing analysis revealed an analogous downregulation of these factors in animal tumor xenografts. These data demonstrate miR-644a mediated fine-tuning of oncogenesis, stimulating pathways and resultant potentiation of enzalutamide therapy in CRPC patients. SIGNIFICANCE: This study demonstrates that miR-644a therapeutically influences the CRPC tumor microenvironment by suppressing androgen signaling and additional genes involved in metabolism, proliferation, Warburg effect, and EMT, to potentiate the enzalutamide therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/8/1844/F1.large.jpg.

Abstract P5-04-20: Targeting CDK4/6 and IGF1R or insulin receptor synergistically inhibits growth of endocrine sensitive and endocrine resistant breast cancers

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.

TROP-2 exhibits tumor suppressive functions in cervical cancer by dual inhibition of IGF-1R and ALK signaling

ObjectiveInactivation of tumor suppressor genes promotes initiation and progression of cervical cancer. This study aims to investigate the tumor suppressive effects of TROP-2 in cervical cancer cells and to explain the underlying mechanisms. MethodsThe tumor suppressive functions of TROP-2 in cervical cancer cells were examined by in vitro and in vivo tumorigenic functional assays. Downstream factors of TROP-2 were screened using Human Phospho-Receptor Tyrosine Kinase Array. Small molecule inhibitors were applied to HeLa cells to test the TROP-2 effects on the oncogenicity of IGF-1R and ALK. Protein interactions between TROP-2 and the ligands of IGF-1R and ALK were detected via immunoprecipitation assay and protein-protein affinity prediction. ResultsIn vitro and in vivo functional assays showed that overexpression of TROP-2 significantly inhibited the oncogenicity of cervical cancer cells; while knockdown of TROP-2 exhibited opposite effects. Human Phospho-Receptor Tyrosine Kinase Array showed that the activity of IGF-1R and ALK was stimulated by TROP-2 knockdown. Small molecule inhibitors AG1024 targeting IGF-1R and Crizotinib targeting ALK were treated to HeLa cells with and without TROP-2 overexpression, and results from cell viability and migration assays indicated that the oncogenicity of vector-transfected cells was repressed to a greater extent by the inhibition of either IGF-1R or ALK than that of the TROP-2-overexpressed cells. Immunoprecipitation assay and protein-protein affinity prediction suggested protein interactions between TROP-2 and the ligands of IGF-1R and ALK. ConclusionsCollectively, our results support that TROP-2 exhibits tumor suppressor functions in cervical cancer through inhibiting the activity of IGF-1R and ALK.

MiRNA-99a can regulate proliferation and apoptosis of human granulosa cells via targeting IGF-1R in polycystic ovary syndrome.

We aimed to evaluate the regulation of miR-99a to the biological functions of granulosa cells in polycystic ovary syndrome (PCOS) via targeting IGF-1R. We collected aspirated follicular fluid in both patients with and without PCOS. Granulosa cells (GCs) were isolated through Percoll differential centrifugation to detect both miR-99a and IGF-1R expressions. We further transfected COV434 cells with miR-99a mimics to establish a miRNA-99a (miR-99a) overexpression model. We explored the regulation of miR-99a to the proliferation and apoptosis of human GCs via IGF-1R in COV434. The effect of different insulin concentrations on miR-99a expression was also evaluated. MiR-99a was significantly downregulated while IGF-1R was upregulated in patients with PCOS. MiR-99a can regulate IGF-1R on a post-transcriptional level. After transfection of miR-99a mimics, the proliferation rate was decreased and apoptosis rate was increased significantly in COV434. Exogenous insulin-like growth factor 1 (IGF-1) treatment could reverse the effect of miR-99a. MiR-99a was negatively and dose-dependently regulated by insulin in vitro. MiR-99a expression was downregulated in patients with PCOS, the degree of which may be closely related to insulin resistance and hyperinsulinemia. MiR-99a could attenuate proliferation and promote apoptosis of human GCs through targeting IGF-1R, which could partly explain the abnormal folliculogenesis in PCOS.

443TiP - First-in-human trial design for W0101: A first-in-class antibody-drug conjugate targeting IGF-1R and identification of the target patient population

443TiP - First-in-human trial design for W0101: A first-in-class antibody-drug conjugate targeting IGF-1R and identification of the target patient population

15 Identification of new combination therapies for lung cancer tumours harbouring KRAS mutations

IntroductionOncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. We have previously shown that NSCLC cells harbouring KRAS mutations are more sensitive than their wild-type counterparts to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors.Material and methodsIn order to identify complementary targets for the improvement of IGF1R and/or MEK targeting therapies, we performed a whole-genome shRNA screen with KRAS-mutant NSCLC cells. Validation was performed using shRNAs and small molecule inhibitors in a panel of NSCLC cell lines. In vivo efficacy of drug combinations was measured in mouse models of Kras-induced NSCLC using CT scanning.Results and discussionsThe list of sensitizers to IGF1R inhibitors included several genes encoding components of the mTOR pathway. Viability assays in a panel of lung cancer cell lines confirmed that combining IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS-mutant NSCLC cells. Mechanistic investigations demonstrated that IGF1R inhibitors blocked reactivation of the PI3K pathway induced by mTOR inhibition, resulting in a robust suppression of PI3K and mTORC1 signalling. Addition of a MEK inhibitor to the combination produced a more profound and durable suppression of cell proliferation and a stronger induction of apoptosis by inhibiting the main downstream pathways controlled by KRAS. Notably, the inhibition of these signalling pathways was stronger in KRAS-mutant cells than in wild-type cells. In order to achieve strong downstream pathway inhibition even more specifically in KRAS-mutant cells, we used the KRAS-G12C mutant inhibitor, ARS-1620. Interestingly, addition of mTOR and IGF1R inhibitors vastly increases effectiveness of the KRAS-G12C inhibitor.Finally, we validated the drug combinations in mouse models of KRAS-induced NSCLC. Results showed that combined mTOR, IGF1R and MEK inhibition produced a marked tumour regression in a NSCLC mouse model driven by mutant Kras and p53 loss-of-function and also in urethane-induced lung tumours.ConclusionWe have demonstrated that a profound inhibition of the main pathways downstream KRAS is needed to achieve a durable suppression of cell viability in NSCLC cells harbouring KRAS mutations. These findings suggest potential novel therapeutic strategies for KRAS mutant NSCLC tumours.

Abstract 2987: Identification of new combination therapies for lung tumors harboring KRAS mutations

Abstract Oncogenic mutations in KRAS are frequent in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to existing therapies. Thus, new therapeutic strategies are needed to treat these tumors. We have previously shown that NSCLC cells harboring KRAS mutations are more sensitive to inhibition of KRAS downstream effectors MEK and RAF and to treatment with IGF1R inhibitors than their wild-type counterparts. In order to identify complementary targets for the improvement of IGF1R and/or MEK targeting therapies, we performed a whole-genome shRNA screen in KRAS-mutant NSCLC cells. Interestingly, the list of sensitizers to IGF1R inhibitors included several genes encoding components of the mTOR pathway. Viability assays in a panel of lung cancer cell lines confirmed that combinations of IGF1R inhibitors with mTOR inhibitors, both rapalogs and kinase inhibitors, resulted in a synergistic anti-proliferative effect in KRAS-mutant NSCLC cells. Mechanistic investigations demonstrated some differences between the effects of rapalogs and mTOR kinase inhibitors although, in both cases, mTOR inhibitors increased IGF1R and AKT phosphorylation. Combination with IGF1R inhibitors blocked the reactivation of the PI3K pathway resulting in a robust suppression of PI3K and mTORC1 signaling. Notably, the inhibition of these signaling pathways was stronger in KRAS-mutant cells than in wild-type cells. Addition of a MEK inhibitor to the combination produced a more profound and durable suppression of cell proliferation and a stronger induction of apoptosis. Finally, we validated the different drug combinations in mouse models of KRAS-induced NSCLC. Results showed that the three-drug combination produced a marked tumor regression in a NSCLC mouse model driven by mutant Kras and p53 loss-of-function and also in urethane-induced lung tumors. These findings suggest potential novel therapeutic strategies for NSCLC tumors harboring KRAS mutations. Citation Format: Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Febe van Maldegem, Stuart Horswell, David Hancock, Julian Downward. Identification of new combination therapies for lung tumors harboring KRAS mutations [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 2987.