Inhibitors In Preclinical Models Research Articles

Dual Angiotensin Receptor-Neprilysin Inhibition With Sacubitril/Valsartan Attenuates Systolic Dysfunction in Experimental Doxorubicin-Induced Cardiotoxicity

BackgroundDoxorubicin (DOX) induces cardiotoxicity in part by activation of matrix metalloproteinases (MMPs). Sacubitril/valsartan (Sac/Val) exerts additive cardioprotective actions over renin-angiotensin-aldosterone inhibitors in preclinical models of myocardial infarction and in heart failure patients. We hypothesized that Sac/Val would be more cardioprotective than Val in a rodent model of progressive DOX-induced cardiotoxicity, and this benefit would be associated with modulation of MMP activation. ObjectivesWe sought to investigate the efficacy of Sac/Val for the treatment of anthracycline-induced cardiotoxicity. MethodsMale Wistar rats received DOX intraperitoneally (15 mg/kg cumulative) or saline over 3 weeks. Following the first treatment, control animals were gavaged daily with water (n = 25), while DOX-treated animals were gavaged daily with water (n = 25), Val (31 mg/kg; n = 25) or Sac/Val (68 mg/kg; n = 25) for either 4 or 6 weeks. Echocardiography was performed at baseline, and 4 and 6 weeks after DOX initiation. In addition, myocardial MMP activity was assessed with 99mTc-RP805, and cardiotoxicity severity was assessed by histology at these time points in a subgroup of animals. ResultsLeft ventricular ejection fraction decreased by 10% at 6 weeks in DOX and DOX + Val rats (both p < 0.05), while this reduction was attenuated in DOX + Sac/Val rats. MMP activity was increased at 6 weeks by 76% in DOX-alone rats, and tended to increase in DOX + Val rats (36%; p = 0.051) but was similar in DOX + Sac/Val rats as compared with time-matched control animals. Both therapies attenuated histological evidence of cellular toxicity and fibrosis (p < 0.05). ConclusionsSac/Val offers greater protection against left ventricular remodeling and dysfunction compared with standard angiotensin receptor blocker therapy in a rodent model of progressive DOX-induced cardiotoxicity.

Discovery of Potent, Highly Selective, and In Vivo Efficacious, Allosteric MALT1 Inhibitors by Iterative Scaffold Morphing.

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.

Phase I Study of Ixazomib with Conventional Chemotherapy in the Treatment of Acute Myeloid Leukemia in Older Adults

Phase I Study of Ixazomib with Conventional Chemotherapy in the Treatment of Acute Myeloid Leukemia in Older Adults

RX-5902, a novel \u03b2-catenin modulator, potentiates the efficacy of immune checkpoint inhibitors in preclinical models of triple-negative breast Cancer

BackgroundTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear β-catenin signaling. The purpose of this study was to evaluate the ability of β-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC.MethodsTreatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2nullIl2rγnullSIRPαNOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells.ResultsThe addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p < 0.05). In the RX-5902/nivolumab combination group, there was a significant increase in the percentage of CD4+ T cells in TILs and increased systemic granzyme B production (p < 0.01).ConclusionsConclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

CDK 4/6 Inhibition Overcomes Acquired and Inherent Resistance to PI3Kα Inhibition in Pre-Clinical Models of Head and Neck Squamous Cell Carcinoma.

Activating alterations in PIK3CA, the gene coding for the catalytic subunit of phosphoinositide-3-kinase (PI3K), are prevalent in head and neck squamous cell carcinoma (HNSCC) and thought to be one of the main drivers of these tumors. However, early clinical trials on PI3K inhibitors (PI3Ki) have been disappointing due to the limited durability of the activity of these drugs. To investigate the resistance mechanisms to PI3Ki and attempt to overcome them, we conducted a molecular-based study using both HNSCC cell lines and patient-derived xenografts (PDXs). We sought to simulate and dissect the molecular pathways that come into play in PIK3CA-altered HNSCC treated with isoform-specific PI3Ki (BYL719, GDC0032). In vitro assays of cell viability and protein expression indicate that activation of the mTOR and cyclin D1 pathways is associated with resistance to PI3Ki. Specifically, in BYL719-resistant cells, BYL719 treatment did not induce pS6 and pRB inhibition as detected in BYL719-sensitive cells. By combining PI3Ki with either mammalian target of rapamycin complex 1 (mTORC1) or cyclin D1 kinase (CDK) 4/6 specific inhibitors (RAD001 and abemaciclib, respectively), we were able to overcome the acquired resistance. Furthermore, we found that PI3Ki and CDK 4/6 inhibitors have a synergistic anti-tumor effect when combined in human papillomavirus (HPV)-negative/PIK3CA-WT tumors. These findings provide a rationale for combining PI3Ki and CDK 4/6 inhibitors to enhance anti-tumor efficacy in HNSCC patients.

Microglial mTOR is Neuronal Protective and Antiepileptogenic in the Pilocarpine Model of Temporal Lobe Epilepsy.

Excessive activation of mammalian target of rapamycin (mTOR) signaling is epileptogenic in genetic epilepsy. However, the exact role of microglial mTOR in acquired epilepsy remains to be clarified. In the present study, we found that mTOR is strongly activated in microglia following excitatory injury elicited by status epilepticus. To determine the role of microglial mTOR signaling in excitatory injury and epileptogenesis, we generated mice with restrictive deletion of mTOR in microglia. Both male and female mice were used in the present study. We found that mTOR-deficient microglia lost their typical proliferative and inflammatory responses to excitatory injury, whereas the proliferation of astrocytes was preserved. In addition, mTOR-deficient microglia did not effectively engulf injured/dying neurons. More importantly, microglial mTOR-deficient mice displayed increased neuronal loss and developed more severe spontaneous seizures. These findings suggest that microglial mTOR plays a protective role in mitigating neuronal loss and attenuating epileptogenesis in the excitatory injury model of epilepsy.SIGNIFICANCE STATEMENT The mammalian target of rapamycin (mTOR) pathway is strongly implicated in epilepsy. However, the effect of mTOR inhibitors in preclinical models of acquired epilepsy is inconsistent. The broad presence of mTOR signaling in various brain cells could prevent mTOR inhibitors from achieving a net therapeutic effect. This conundrum has spurred further investigation of the cell type-specific effects of mTOR signaling in the CNS. We found that activation of microglial mTOR is antiepileptogenic. Thus, microglial mTOR activation represents a novel antiepileptogenic route that appears to parallel the proepileptogenic route of neuronal mTOR activation. This may explain why the net effect of mTOR inhibitors is paradoxical in the acquired models of epilepsy. Our findings could better guide the use of mTOR inhibitors in preventing acquired epilepsy.

Abstract 5357: Total abdominal ultra-rapid FLASH irradiation enhances the efficacy of PD-1 inhibition in preclinical models of ovarian cancer

Abstract Objective: Ovarian cancer often presents with advanced stage disease. Despite surgery and chemotherapy, recurrence remains inevitable and prognosis poor. Advances in immunotherapy including programmed cell death-1 (PD-1) inhibition show promise, however clinical trials show modest response rates as a single agent therapy in ovarian cancer patients. Radiation therapy is an attractive strategy to enhance the efficacy of immune checkpoint blockade in ovarian cancer due to its ability to increase tumor-infiltrating lymphocytes and enhance immune activation. While ovarian cancer is known to be radiosensitive, total abdominal radiation is not routinely utilized due to high abdominopelvic toxicity. We have recently shown that ultra-high dose rate FLASH irradiation reduces intestinal radiation-induced toxicity. Importantly, we demonstrated efficient tumor control in preclinical models of ovarian cancer peritoneal metastasis. Here we sought to determine the efficacy and safety of FLASH irradiation combined with PD-1 inhibition in preclinical models of ovarian cancer. Methods: Female C57BL/6 mice were intraperitoneally inoculated with ID8-Asc or UPK10 ovarian cancer cells. Total abdominal FLASH irradiation at 14 Gy was administered 10 days post-inoculation. Anti-PD-1 and isotype control antibodies were injected on days 7, 10, and 13 post-inoculation. Mice were sacrificed 22-27 days post-inoculation for tumor burden and immune cell analysis. Stool count, stool weight, complete blood count, and survival were evaluated to compare acute and chronic toxicity as well as efficacy in the treatment arms. Results: Four cohorts of mice were analyzed: Isotype control antibody (IgG), anti-PD-1 (aPD-1), FLASH + IgG, and FLASH + aPD-1. In the ID8-Asc model, irradiated mice showed a 44% decrease in solid stool production with subsequent recovery to baseline by 14 days post-treatment. There was no evidence of hematopoietic toxicity in any of the treatment arms. Survival analysis is ongoing. Exploratory necropsy demonstrated significant tumor burden control in the FLASH cohorts, and in particular, a 25-fold decrease in tumor number in the FLASH + aPD-1 cohort compared to control (mean tumor weight 1.82 mg vs. 357.2 mg, respectively). FLASH + aPD-1 increased intratumoral CD4+ and CD8+ T cells and correlated with enhanced tumor control compared to FLASH or aPD-1 single agent treatments. Similar results were observed in the UPK10 model. Conclusion: Combination FLASH and PD-1 inhibition enhanced tumor control and recruitment of tumor infiltrating lymphocytes compared to IgG, aPD-1, and FLASH treatments alone, while demonstrating safety and tolerability. Our data identify a new opportunity for FLASH and checkpoint inhibition in the treatment of ovarian cancer. Citation Format: Stephanie Chow, Joshua T. Eggold, Karen Levy, Jinghui Wang, Rakesh Manjappa, Dylan Y. Breitkreutz, Amy S. Yu, Karl Bush, Oliver Dorigo, Billy W. Loo, Erinn B. Rankin. Total abdominal ultra-rapid FLASH irradiation enhances the efficacy of PD-1 inhibition in preclinical models of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5357.

Abstract 5179: Isatuximab based combinations induce potent tumor growth inhibition in pre-clinical models of multiple myeloma and acute lymphocytic leukemia

Abstract CD38 is highly expressed in many hematological malignancies such as multiple myeloma (MM) and acute lymphocytic leukemia (ALL) and can be targeted by antibody-based therapy. Isatuximab, an anti-CD38 IgG1 monoclonal antibody has promising clinical activity in relapsed/refractory multiple myeloma patients. It induces tumor cells death via Fc-mediated cytotoxic effects, direct cancer cell killing, and inhibition of CD38 enzymatic activity. We investigated the mechanisms of action (MoA) of isatuximab against MM and ALL in vitro and evaluated its in vivo pharmacological potency in combination with standard therapies for these two hematologic malignancies. Isatuximab preferentially induced robust antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) in both MM and ALL cell lines. Cancer cells with particularly high level of CD38 were also susceptible to apoptosis or CDC induced by isatuximab. In vitro, isatuximab displayed time- and dose-dependent inhibitory effect on CD38 enzymatic activity shown in LP-1 myeloma cells, suggesting that it could alleviate immunosuppressive effects of CD38-mediated adenosine synthesis in the tumor microenvironment. In vivo efficacy studies were conducted in mouse xenograft models. The in vivo efficacy of isatuximab in combination with the immunomodulatory drugs (IMiDs) pomalidomide or lenalidomide that are standard of care in MM was evaluated. Each combination increased the inhibition of tumor growth (T/C) compared with isatuximab or IMiDs treatment alone. Combination of isatuximab with either bortezomib or carfilzomib, two proteasome inhibitors that are widely used in MM, significantly improved their anti-tumor activity versus each agent alone. Isatuximab also improved tumor growth inhibition mediated by alkylating agent melphalan. Similarly, isatuximab enhanced the activity of different chemotherapies in an ALL tumor model. Its combination with vincristine, cytarabine or cyclophosphamide enhanced antitumor activity translating into a higher number of tumor-free survivors (TFS). These results document the unique MoA of isatuximab, primarily through ADCC and ADCP, and its robust antitumor activity as a single agent and in combination with standard of care treatments used in these hematological malignancies. ChemotherapyMM or T-ALL xenograft modelT/C IsatuximabT/C ChemotherapyT/C combinationPomalidomideMOLP-862%54%34%LenalidomideRPMI-822647%76%24%BortezomibNCI-H92928%26%0 %carfilzomibNCI-H92968%44%6%MelphalanLP-167%13%8%VincristineDND4141% no TFS0% no TFS0% 5/6 TFSAra-CDND4141% no TFS0% 3/6 TFS0% 6/6 TFScyclophosphamideDND4110% 4/7 TFS0% 4/7 TFS0% 7/7 TFS Citation Format: Chen Zhu, Celine Nicolazzi, Helgi Van de Velde, Dmitri Wiederschain, Sukhvinder Sidhu, Marielle Chiron. Isatuximab based combinations induce potent tumor growth inhibition in pre-clinical models of multiple myeloma and acute lymphocytic leukemia [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 5179.

Abstract 2404: MDM2 inhibitor Idasanutlin potentiate therapeutic agents in Diffuse Large B-Cell Lymphoma by targeting MDM2 and XIAP

Abstract Purpose: In patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), resistance to rituximab confers chemotherapy-resistance and poor response to salvage regimens. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets, but is known best as the major negative regulator of the tumor suppressor, p53. Recently, we demonstrated that MX69, a MDM2 inhibitor, re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro by blocking the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, thus establishing the feasibility of this approach for overcoming rituximab and chemotherapy resistance in DLBCL. We now report the activity of Idasanutlin, an investigational Nutlin-family MDM2 antagonist, in pre-clinical models of R-chemo resistant B-cell lymphoma. Methods: We used a panel of rituximab sensitive and R-chemo resistant B-cell lymphoma cell lines representing activated B-cell-like (ABC) DLBCL (TMD8, U2932); germinal center B-cell like (GCB) DLBCL (RL, RL 4RH, OCILy2); double hit DLBCL (DHL4, DHL6, DOHH2, VAL, ROS50), and Burkitt's lymphoma (Raji and Raji 4RH). Cells were exposed to escalating doses of Idasanutlin as single agent and in combination with various chemotherapeutic agents and small molecule inhibitors for 24, 48 and 72 hrs. Differences in cell viability were evaluated by PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 followed by flow cytometric analysis. Western blot was used to detect changes in MDM2, XIAP and PUMA expression. Results: In vitro exposure to Idasanutlin demonstrated dose- and time-dependent cell death in DLBCL cell lines, IC50 ranged from 0.7uM to 63.07uM at 48h. Idasanutlin at the dose of 1uM was able to disrupt mitochondria and lowered the mitochondrial membrane potential in both ABC and GCB cell lines at 48 h. Idasanutlin reduced the expression of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP expression was reduced in VAL and DHL4. PUMA, the downstream product of p53 activation, was increased after idasanutlin exposure in all cell lines. Idasanutlin demonstrated significant synergy with chemotherapeutic reagents (etoposide and vincristine), proteasome inhibitors (carfilzomib and ixazomib), Bcl-2 inhibitor venetoclax and Bruton tyrosine kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed promising cell-of-origin agnostic anti-tumor activity as a single agent and in combination with several small molecule inhibitors in pre-clinical models of DLBCL. It decreased MDM2 and XIAP protein level and induced PUMA expression. Its unique mechanism of action through p53 modulation makes it an attractive partner for use in a variety of clinical settings in B-cell lymphomas. Citation Format: Juan J. Gu, Samuel Thompson, Cory Mavis, Pallawi Torka, Francisco Hernandez-Ilizaliturri. MDM2 inhibitor Idasanutlin potentiate therapeutic agents in Diffuse Large B-Cell Lymphoma by targeting MDM2 and XIAP [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 2404.

Abstract LB-113: Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers

Abstract The RAS and PI3K/mTOR signaling pathways function downstream of receptor tyrosine kinases (RTK), and are hyperactivated in a wide range of human cancers. Several points of cross-talk and intersecting feedback mechanisms have also been described, in which inhibition of one pathway node leads to activation of the parallel signaling pathway, thereby decreasing the effectiveness of single-pathway targeted therapies. Concomitant inhibition of both pathways is clearly effective in RAS-driven cancers as demonstrated via genetic means and by the combination of pharmacologic inhibitors of MEK and PI3 kinase in a variety of preclinical models (Engelman 2008, Collisson 2012, Eser 2013) However, the clinical translation of this combination has been largely unsuccessful, primarily due to development of dose-limiting toxicities that prohibit the achievement of optimal therapeutic drug concentrations. Recent advances in the treatment paradigm for RAS mutant non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) include several first-in-class covalent inhibitors of KRASG12C (e.g., AMG510, MRTX849) which have demonstrated a favorable safety profile along with early promising anti-tumor activity in patients with advanced cancers harboring a KRASG12C mutation. Drug-anchored CRISPR screens and combination experiments have identified mTOR signaling as a key mediator that limits therapeutic response to KRASG12C inhibition (Molina-Arcas 2019, Hallin 2019). We have developed a novel class of selective mTORC1 inhibitors, termed ‘bi-steric', that covalently links rapamycin analogs with a mTOR kinase active-site inhibitor, and interacts with both the ATP- and FKBP12/FRB-binding sites of mTORC1. These compounds exhibit potent and selective (&amp;gt;10-fold) inhibition of mTORC1 over mTORC2, durably suppress S6K and 4EBP1 phosphorylation, and induce growth suppression and apoptosis in multiple cancer cell lines. In preclinical tests, these mTORC-1 selective inhibitors do not induce hyperglycemia and are predicted to have reduced mTORC2-mediated toxicities. We tested the combinatorial activity of these mTOR inhibitors and covalent KRASG12C inhibitors in preclinical models of two KRASG12C mutant tumor types predicted to have limited sensitivity to KRASG12C inhibitor monotherapy: a) STK11 deficient NSCLC wherein activation of mTORC1 signaling is also an oncogenic driver (~20% co-occurrence in KRAS-mutant NSCLC, Scheffler 2018) and b) colorectal cancer wherein RTK activation is thought to co-activate PI3 kinase signaling (Ebi, 2011). In each case, we showed combinatorial activity manifest as decreased tumor cell viability in vitro and tumor regressions in vivo. In contrast, single agents exhibited cytostatic effects in vitro and modest effects on tumor growth in vivo. Our data demonstrate that the combination of KRASG12C and bi-steric mTORC1 selective inhibitors elicits marked anti-tumor activity at well tolerated doses in vivo in models of cancer patient populations with current unmet medical need. The mechanistic bases for these combinatorial effects are currently under investigation. Our preclinical results reinforce the importance of mTORC1 as a concurrent target in genetically defined RAS-mutant cancers, and showcase the therapeutic potential of a novel and selective bi-steric mTORC1 inhibitor, currently in IND-enabling studies. Citation Format: Yu Chi Yang, Christopher Schulze, Jingjing Jiang, Denise F. Reyes, Tiffany Choy, Tram Nguyen, Zhican Wang, Dong Lee, Robert J. Nichols, Zhengping Wang, Jacqueline A. Smith, Steve M. Kelsey, Mallika Singh. Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers [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 LB-113.

Abstract 6645: Anti-EGF antibodies significantly improve the activity of RET, BRAF, MEK and PI3K kinase inhibitors in preclinical models

Abstract Background: We have recently shown that antibodies generated by vaccination (anti-EGF VacAbs) potentiate the effects of tyrosine kinase inhibitors (TKIs) in epidermal growth factor receptor mutant (EGFR-mut) cell lines (1) and a Phase I/II clinical trial of an anti-EGF vaccine in combination with afatinib has been initiated. In this study we aimed to determine the efficacy of anti-EGF VacAbs to improve the antitumor activity of RET, BRAF, MEK and PI3K inhibitors in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines. Methods: Cell lines with RET translocations (LC-2/ad) and BRAF, KRAS and PIK3CA mutations (HT29, DLD1, LS174T and H508) were used. Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant human EGF. Cell lines were treated with anti-EGF VacAbs alone and in combination with RET, BRAF, MEK and PI3K inhibitors. Cell viability was determined by MTT, cell cycle was analyzed by flow cytometry, changes of total and phosphorylated proteins by Western blot and emergence of resistance by direct microscopic examination in low density cultures. Results: Anti-EGF VacAbs suppressed EGF-induced cell proliferation and blocked EGFR transduction signaling pathway in all cell lines tested. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of BLU667 in LC-2/ad, trametinib and encorafenib in HT29, trametinib in DLD1 and LS174T and trametinib, taselisib, alpelisib and copanlisib in H508 cells. In these cell lines, anti-EGF VacAbs in combination with kinase inhibitors suppressed EGFR, Akt and Erk 1/2 phosphorylation. Cell cycle experiments revealed that anti-EGF VacAbs significantly increased the antiproliferative effects of the kinase inhibitors, measured as the percentage of cells in G2+M. Finally, the addition of the anti-EGF VacAbs to the culture medium significantly delayed the emergence resistant clones to trametinib in DLD1 cells. Conclusions: Anti-EGF VacAbs potentiate the antitumor effects of RET, MEK, BRAF and PI3K inhibitors in tumor cell lines. Our data provide a rationale for clinical trials testing the combination of anti-EGF VacAbs with kinase inhibitors in RET-translocated, KRAS, BRAF and PIK3CA mutant NSCLC and CRC patients.(1)“Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells” Codony-Servat J, García-Roman S, Molina-Vila MA, et al. J Thorac Oncol. 2018. Citation Format: Silvia Garcia Roman, Jordi Codony Servat, Miguel Angel Molina Vila, Jordi Bertran Alamillo, Monica Garzon, Alejandro Martínez Bueno, Santiago Viteri, María González Cao, Andrés Aguilar, Juan José García, Erik d'Hondt, Rafael Rosell. Anti-EGF antibodies significantly improve the activity of RET, BRAF, MEK and PI3K kinase inhibitors in preclinical models [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 6645.

Abstract 4840: Radiation therapy as a tool to sensitize HR+ breast cancer and mantle cell lymphoma to CDK4/6 inhibitors

Abstract Loss of cell cycle control is a common feature of most (if not all) human cancers. Thus, cell cycle genes that are frequently dysregulated in malignant cells, including CDK4 and CDK6, have attracted considerable attention as targets for the development of novel anticancer agents. This approach has culminated with the FDA approval of selective CDK4/6 inhibitors (e.g. palbociclib) for the treatment of hormone receptor (HR)+ breast cancer (BC). The same inhibitors are currently being tested in patients with a variety of solid and hematological tumors, including mantle cell lymphoma (MCL). However, not all BC and MCL patients achieve durable complete responses on CDK4/6 inhibitors, calling for the development of mechanism-based combination therapies. In this context, focal radiation therapy (RT) stands out as a promising therapeutic partner because both RT and CDK4/6 inhibitors have cytostatic/cytotoxic as well as immunostimulatory effects, but they operate with different mechanisms. We therefore decided to investigate the potential synergy/cooperation between RT and CDK4/6 inhibitors, with a particular focus on administration schedule, to inform the design of clinical trials testing the combination of these therapeutic agents in patients with HR+HER2-BC and MCL. We harnessed palbociclib-sensitive and -resistant MCL cells (MAVER-1 and MAVER-1R) and BC cells (MCF7 and MBA-MB-231) to investigate the ability of RT to synergize with palbociclib in vitro. In addition, we tested these combinations in vivo, in a unique immunocompetent mouse model of mammary carcinogenesis that recapitulates multiple immunobiological features of human HR+HER2-BC. In this model endogenous mammary carcinomas are driven by a slow-release progesterone pellet plus DMBA as an oral carcinogen. We first determined the lowest doses of RT and palbociclib that mediate robust short-term cytostatic/cytotoxic effects in BC and MCL cells. Optimized dose combinations and sequencing experiments pointed to RT followed by palbociclib as the approach with superior therapeutic potential, in vitro. This largely reflects the ability of palbociclib to induce G1 arrest of cells that escaped RT killing during G2/M arrest. In addition, low doses of RT were able to enhance palbociclib sensitivity in MAVER-1R cells. Both RT and palbociclib were active in the endogenous mammary carcinomas model, but failed to mediate disease eradication. In vivo experiments showed that RT preceding CDK4/6 inhibition achieved superior disease control, compared to other sequencing regimens. In conclusion, our results identified an optimal therapeutic sequence to combine RT and CDK4/6 inhibition in preclinical models of BC and MCL, providing a rational for combining stereotactic body RT and palbociclib in a clinical trial for patients with newly diagnosed HR+ oligo-metastatic BC (up to five metastatic lesions). Future directions include the evaluation of the immunostimulatory effects of RT plus palbociclib in the same models of BC and MCL. Supported by SU2C-Ziskin's Award # ZP-6177. Citation Format: Giulia Petroni, Maurizio Di Liberto, Aitziber Buqué Martinez, Selina Chen-Kiang, Lorenzo Galluzzi, Silvia C. Formenti. Radiation therapy as a tool to sensitize HR+ breast cancer and mantle cell lymphoma to CDK4/6 inhibitors [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 4840.

Effect of murine double-minute 2 inhibitors in preclinical models of advanced clear cell carcinomas originating from ovaries and kidneys.

Advanced clear cell carcinomas originating from both ovaries and kidneys with cancerous peritonitis have poor prognoses. Murine double‐minute 2 (MDM2) is a potential therapeutic target for clear cell ovarian carcinomas with WT TP53. Herein, we characterized the antiangiogenic and antitumor effects of the MDM2 inhibitors DS‐3032b and DS‐5272 in 6 clear cell ovarian carcinoma cell lines and 2 clear cell renal carcinoma cell lines, as well as in clear cell ovarian carcinomas s.c. xenograft and ID8 (murine ovarian cancer cells with WT TP53) cancer peritonitis mouse models. In clear cell ovarian carcinoma s.c. xenograft mouse models, DS‐3032b significantly reduced WT TP53 clear cell ovarian carcinoma‐ and clear cell renal carcinoma‐derived tumor volumes. In ID8 mouse models, DS‐5272 significantly inhibited ascites production, reduced body weight, and significantly improved overall survival. Additionally, DS‐5272 reduced the tumor burden of peritoneal dissemination and decreased CD31+ cells in a dose‐dependent manner. Furthermore, DS‐5272 significantly decreased vascular endothelial growth factor concentrations in both sera and ascites. Combined therapy with MDM2 inhibitors and everolimus showed synergistic, and dose‐reduction potential, for clear cell carcinoma treatment. Our findings suggest that MDM2 inhibitors represent promising molecular targeted therapy for clear cell carcinomas, thereby warranting further studies to evaluate the efficacy and safety of dual MDM2/mTOR inhibitors in clear cell carcinoma patients.

Design and screening of FAK, CDK 4/6 dual inhibitors by pharmacophore model, molecular docking, and molecular dynamics simulation

Focal adhesion kinase (FAK) is one kind of tyrosine kinases that modulates integrin and growth factor signaling pathways, which is a promising therapeutic target because of involving in the migration, proliferation and survival of cancer cell. Overexpression and amplification of cyclin-dependent kinase 4/6 (CDK4/6) occur in many cancers and may be the cause of resistance to CDK4/6 inhibitors in preclinical models. The latest research shows that the combination of FAK and CDK4/6 can be dually targeted to enhance the antitumor effects. In this study, FAK and CDK4/6 dual target inhibitors were designed by computer-aided drug design. Seven million molecules were screened by the pharmacophore model and molecular docking. Finally, 6 compounds were obtained. Molecular dynamics simulation of compound 1, 2 and 3 showed that it has good binding stability to both receptors. According to the binding modes of compound 1 with two receptors, corresponding modifications were made, and 7 novel designed compounds were obtained. The docking energy of these novel designed compounds were lower than that of compound 1, and they can be tested in future. Communicated by Ramaswamy H. Sarma

CXCR4 Inhibition Enhances Efficacy of FLT3 Inhibitors in FLT3-Mutated AML Augmented by Suppressed TGF-b Signaling.

Given the proven importance of the CXCL12/CXCR4 axis in the stroma–acute myeloid leukemia (AML) interactions and the rapid emergence of resistance to FLT3 inhibitors, we investigated the efficacy and safety of a novel CXCR4 inhibitor, LY2510924, in combination with FLT3 inhibitors in preclinical models of AML with FLT3-ITD mutations (FLT3-ITD-AML). Quizartinib, a potent FLT3 inhibitor, induced apoptosis in FLT3-ITD-AML, while LY2510924 blocked surface CXCR4 without inducing apoptosis. LY2510924 significantly reversed stroma-mediated resistance against quizartinib mainly through the MAPK pathway. In mice with established FLT3-ITD-AML, LY2510924 induced durable mobilization and differentiation of leukemia cells, resulting in enhanced anti-leukemia effects when combined with quizartinib, whereas transient effects were seen on non-leukemic blood cells in immune-competent mice. Sequencing of the transcriptome of the leukemic cells surviving in vivo treatment with quizartinib and LY2510924 revealed that genes related to TGF-β signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF-β in stromal cells or TGF-β-receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF-β signaling.

Venetoclax combines synergistically with FLT3 inhibition to effectively target leukemic cells in FLT3-ITD+ acute myeloid leukemia models.

FLT3 internal tandem duplication (FLT3-ITD) mutations account for approximately 25% of adult acute myeloid leukemia (AML) cases and are associated with poor prognosis. Venetoclax, a selective BCL-2 inhibitor, has limited monotherapy activity in relapsed/refractory AML with no responses observed in a small subset of FLT3-ITD+ patients. Further, FLT3-ITD mutations emerged at relapse following venetoclax monotherapy and combination therapy suggesting a potential mechanism of resistance. Therefore, we investigated the convergence of FLT3-ITD signaling on the BCL-2 family proteins and determined combination activity of venetoclax and FLT3-ITD inhibition in preclinical models. In vivo, venetoclax combined with quizartinib, a potent FLT3 inhibitor, showed greater anti-tumor efficacy and prolonged survival compared to monotherapies. In a patient-derived FLT3-ITD+ xenograft model, cotreatment with venetoclax and quizartinib at clinically relevant doses had greater anti-tumor activity in the tumor microenvironment compared to quizartinib or venetoclax alone. Use of selective BCL-2 family inhibitors further identified a role for BCL-2, BCL-XL and MCL-1 in mediating survival in FLT3-ITD+ cells in vivo and highlighted the need to target all three proteins for greatest anti-tumor activity. Assessment of these combinations in vitro revealed synergistic combination activity for quizartinib and venetoclax but not for quizartinib combined with BCL-XL or MCL-1 inhibition. FLT3-ITD inhibition was shown to indirectly target both BCL-XL and MCL-1 through modulation of protein expression, thereby priming cells toward BCL-2 dependence for survival. These data demonstrate that FLT3-ITD inhibition combined with venetoclax has impressive anti-tumor activity in FLT3-ITD+ AML preclinical models and provides strong mechanistic rational for clinical studies.

Correction: EGFR Exon 20 Insertion Mutations Display Sensitivity to Hsp90 Inhibition in Preclinical Models and Lung Adenocarcinomas.

Purpose: EGFR exon 20 insertions account for up to 10% of all EGFR mutations in lung adenocarcinomas, representing the third most common cluster of mutations. The management of advanced cancers with these mutations remains elusive, without an approved inhibitor. Experimental Design: Preclinical models of a representative set of EGFR exon 20 insertion mutations to evaluate the efficacy of different inhibitors and description of the clinical outcome of an advanced lung cancer. Results: We show that select first-, second-, and third-generation EGFR inhibitors are unable to deter common EGFR exon 20 insertion mutants in concentrations that spare the wild-type kinase. Nonetheless, EGFR exon 20 insertion mutants associate with the Hsp90 chaperone system. We exploit this vulnerability to show that the nongeldanamycin Hsp90 inhibitor luminespib (formerly AUY922) degrades EGFR exon 20 mutations, downstream targets, and induces apoptosis. In addition, a patient whose EGFR inhibitor–insensitive lung adenocarcinoma harbored an EGFR exon 20 insertion mutation had a confirmed radiographic response to luminespib. Conclusions: The report confirms that EGFR exon 20 mutations are dependent on Hsp90 and are readily inhibited by the Hsp90 inhibitor luminespib; a treatment strategy that has been pursued in a confirmatory clinical trial (NCT01854034) for this group of lung adenocarcinomas that currently represent an unmet clinical need in precision oncology.

Bromodomain inhibitors revisited

Drug Discovery Bromodomain and extraterminal domain (BET) proteins contribute to the pathogenesis of cancer and immune diseases through their effects on transcriptional regulation. BET proteins contain two nearly identical bromodomains, BD1 and BD2, structural modules that have attracted great interest as targets for drug development. First-generation drugs that inhibited both BD1 and BD2 showed promising therapeutic activity in preclinical models but proved to be less efficacious in clinical trials. Gilan et al. took a different approach and designed drugs that selectively inhibited BD1 or BD2 (see the Perspective by Filippakopoulos and Knapp). They found that BD1 and BD2 inhibitors altered gene expression in different ways and that BD2 inhibitors had greater therapeutic activity than BD1 inhibitors in preclinical models of inflammation and autoimmune disease. Science , this issue p. [387][1]; see also p. [367][2] [1]: /lookup/doi/10.1126/science.aaz8455 [2]: /lookup/doi/10.1126/science.abb5060

Targeting Apoptosis in Acute Myeloid Leukemia: Current Status and Future Directions of BCL-2 Inhibition with Venetoclax and Beyond.

Acute myeloid leukemia (AML) is a disease of the hematopoietic system that remains a therapeutic challenge despite advances in our understanding of the underlying cancer biology over the past decade. Recent developments in molecular targeting have shown promising results in treating leukemia, paving the way for novel treatment strategies. The discovery of drugs that promote apoptosis in leukemic cells has translated to encouraging activity in clinical trials. B-cell lymphoma (BCL)-2 inhibition has been at the center of drug development efforts to target apoptosis in AML. Remarkable clinical success with venetoclax has revolutionized the ways we treat hematological malignancies. Several landmark trials have demonstrated the potent antitumor activity of venetoclax, and it is now frequently combined with traditional cytotoxic agents to treat AML. However, resistance to BCL-2 inhibition is emerging, and alternative strategies to address resistance mechanisms have become an important focus of research. A number of clinical trials are now underway to investigate a plurality of novel agents that were shown to overcome resistance to BCL-2 inhibition in preclinical models. Some of the most promising data come from studies on drugs that downregulate myeloid cell leukemia (MCL)-1, such as cyclin-dependent kinases (CDK) inhibitors. Furthermore, innovative approaches to target apoptosis via extrinsic pathways and p53 regulation have added new cytotoxic agents to the arsenal, including drugs that inhibit inhibitor of apoptosis protein (IAP) family proteins and murine double minute 2 (MDM2). This review provides a perspective on past and current treatment strategies harnessing various mechanisms of apoptosis to target AML and highlights some important promising treatment combinations in development.

30P Assessing the potential role of RICTOR expression as predictive factor of response to PI3K/mTOR pathway inhibitors in preclinical models of squamous cell lung cancer

30P Assessing the potential role of RICTOR expression as predictive factor of response to PI3K/mTOR pathway inhibitors in preclinical models of squamous cell lung cancer