Greater Tumor Growth Inhibition Research Articles

Abstract 4548: SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity

Abstract CD112R (PVRIG) is a recently identified inhibitory receptor of the PVR gene family, which includes TIGIT, CD96, and CD226, that is expressed on NK cells and T cells. CD112R suppresses immune cell activation through its association with the cell adhesion molecule CD112 (PVRL2), a ligand that it competes for with the activating receptor CD226. CD112 binding to CD112R induces downstream signaling via recruitment of SHP-1, SHP-2, and SHIP-1 phosphatases to the ITIM in the cytoplasmic tail, resulting in dampened effector cell activation. As CD112 is commonly upregulated on cancer cells and is also expressed on tumor infiltrating myeloid cells, it was hypothesized that disruption of the CD112:CD112R interaction would promote anti-tumor immune responses. Phenotypic screening of a panel of antibodies generated against CD112R led to the identification of SRF813, a high affinity, fully human CD112R antibody that blocks the interaction of CD112R with CD112. To explore the therapeutic potential of CD112R blockade, the ability of SRF813 to increase immune cell activation was evaluated in human peripheral blood mononuclear cells. In vitro assays showed that SRF813 treatment enhanced NK cell activation in response to multiple tumor cell lines. This activation was characterized by upregulation of several cell surface markers, including 4-1BB and ICAM-1, in addition to increased cytokine production and degranulation. The activity of SRF813 was highly dependent on the antibody isotype and Fc receptor engagement. Mechanistically, the enhanced immunologic responses observed in NK cells treated with SRF813 could be reversed in the presence of a CD226 blocking antibody, confirming the role of CD112R in regulating CD226 mediated signaling. Blockade of CD112R in mouse syngeneic tumor models using a murine surrogate of SRF813 demonstrated reduced tumor growth with an accompanying increase in TIL activation following treatment. In the CT-26 tumor model, anti-CD112R treatment resulted in complete tumor regression in a subset of treated mice. These mice rapidly rejected tumors upon re-challenge, signifying the development of an immunologic anti-tumor memory response in these animals. Additional depletion studies highlighted that the anti-tumor efficacy was dependent on both CD8 T cells and NK cells. Moreover, the combination of anti-CD112R with PD-1 blockade led to greater inhibition of tumor growth than either treatment alone. Collectively, these preclinical data demonstrated that CD112R is a negative regulator of immune responses and that CD112R blockade can potentiate anti-tumor responses in cancers that express CD112. Citation Format: Marisella Panduro, Roy M. Dornbrook, Kshama A. Doshi, Jing Hua, Jamie Strand, Vito J. Palombella, Pamela M. Holland, Jonathan A. Hill, James F. Mohan. SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity [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 4548.

Abstract 5497: Transducer array layouts for treating head and neck cancer with TTFields

Abstract Tumor Treating Fields (TTFields) are a novel antimitotic treatment for solid tumors. TTFields utilize low frequency (1-4 V/cm) electric fields in the intermediate frequency range (100-300 kHz), and are currently FDA approved for the treatment of glioblastoma multiforme (GBM) using the Optune device. TTFields are also approved for multi pleural mesothelioma (MPM). The fields are delivered using two pairs of transducer arrays placed on different places on the patient's skin. In order for the treatment to be efficacious, placement should be performed in a manner such that the tumor receives significant dose of TTFields. An effective dose is considered to be at least 1 V/cm in the region of interest. Nevertheless, preclinical studies has shown that higher field values result in greater tumor growth inhibition. When TTFields are delivered, the field distribution is determined by a series of non-intuitive factors, such as the different electric conductivity of the different organs, making intuitive placement inaccurate. Therefore, different parts of the body require specific array layout design. Such case occurs with head and neck cancer. In this work we examined our ability to deliver TTFields to the larynx and its surroundings. Methods We used computer simulations to estimate the field distribution different layouts would provide. The simulations used a computational phantom representing a 34 years old male (DUKE 3.1, ITI'S foundation, Zurich). We placed virtual transducer arrays comprising either 13 or 9 ceramic disks were placed on the model's skin and delivery of TTFields to the model was simulated Sim4Life v4.4 (by ZMT, Zurich). 6 different array layouts were tested. In layouts 1-3, one array comprising 13-disks was placed on the model's scalp while the other 13-disks array was placed on its chest. Forcase 4 a pair of 9-disks arrays was used, one array was placed on the forehead of the model and the other on it'supper chest. In cases 5-6, we placed 9-disks arrays around the patient's neck. We then analyzed the field intensity within a region of interest (ROI) including the larynx and surrounding tissues. Results In cases 1-2 and 4, mean field intensities within the ROI were 3.29 V/cm, 3.46 V/cm and 2.6 V/cm, respectively, well above the clinical threshold of 1 V/cm. Cases 3, 5 and 6 showed to have median field of 1.41, 1.37 and 1.27 V/cm. Though still above the clinical threshold, these field intensities are significantly lower than in the other cases. This, apparently, is due to current flow through the neck muscles behind the ROI. Conclusions This study shows that it is possible to deliver TTFields at therapeutic intensities to the larynx and the surrounding tissues. Thus, TTFields may be a therapeutic option for treating head and neck tumors located in the larynx and the surrounding tissues. Citation Format: Ze'ev Bomzon, Ariel Naveh, Yissachar Abraham. Transducer array layouts for treating head and neck cancer with TTFields [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 5497.

Abstract 553: Antitumor efficacy of a liposomal formulation of irinotecan in preclinical gastric cancer models: Augmenting its response by antiangiogenic agents

Abstract BACKGROUND: Gastric adenocarcinoma (GAC) remains the 3rd most common cause of cancer-related deaths worldwide. Systemic chemotherapy is commonly a fundamental treatment for metastatic GAC that usually leads to a modest patient benefit, resulting in a 5-year survival rate of 31%. A liposomal formulation of irinotecan (nal-IRI) has shown improved pharmacokinetic and drug biodistribution compared with free irinotecan (IRI) in preclinical studies. Angiogenesis plays a crucial role in the progression and metastasis of GAC. We evaluated the therapeutic efficacy of nal-IRI in combination with nintedanib, a tyrosine kinase inhibitor targeting FGFR, PDGFR and VEGFR, and DC101, a monoclonal antibody targeting VEGFR2 in preclinical models of GAC. METHODS: In vitro cell proliferation was evaluated in three GAC cell lines (MKN-45, KATO-III and SNU-5) by WST-1 assay. Protein expression was measured by Western blot analysis in MKN-45 cell lysates. Animal survival studies were performed using two cell lines MKN-45 and KATO-III cells in peritoneal dissemination models in NOD/SCID mice (n=5-7). Tumor growth and pharmacokinetic studies were performed in GAC cell-derived xenografts. Mechanistic evaluation included IHC and Immunoblot analysis in tumor tissues. RESULTS: Animal survival was increased by nal-IRI (>156%) and IRI (>94%) therapy compared with PBS-treated controls. Importantly, nal-IRI led to a >30% extension of animal survival compared with IRI. The addition of antiangiogenic agents nintedanib or DC101 led to a further improvement (range 3-40%) in animal survival caused by nal-IRI and IRI. In GAC cell-derived subcutaneous xenografts, compared to controls, nal-IRI demonstrated greater tumor growth inhibition (92%) than IRI (71%). Here, the combinations of nal-IRI or IRI with antiangiogenic agents exhibited an additive response. Ki67 staining of tumor sections revealed that nal-IRI was most effective in reducing intratumoral proliferation (by 58%), followed by IRI (39%), nintedanib (33%), and DC101 (25%). Combinations of nal-IRI or IRI with antiangiogenic agents demonstrated an additive effect in reducing tumor cell proliferation. As expected, tumor vasculature (assessed by endomucin staining) was reduced by nintedanib (65%) and DC101 (58%), while nal-IRI and IRI showed no effect. Pharmacokinetic studies revealed that nal-IRI increased the retention, circulation time and tumor levels of IRI and its active metabolite SN-38. The addition of nintedanib or DC101 had no effect on plasma or tumor levels of IRI or SN38. In in vitro cell viability assays in mutationally different GAC cells, SN-38 had a dose-dependent growth inhibitory effect that was more pronounced than single-agent IRI. Importantly, combinations of SN-38 or IRI with antiangiogenic agents demonstrated additive effects on cell proliferation inhibition. CONCLUSION: nal-IRI showed greater antitumor efficacy than IRI, and its antitumor effects can be enhanced by antiangiogenic agents suggesting that this combination has potential for improving clinical GAC therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Changhua Zhang, Stephan Klinz, Florence Meyer-Losic, Benjamin Beaufils, Arunthathi Thiagalingam, Roderich E. Schwarz. Antitumor efficacy of a liposomal formulation of irinotecan in preclinical gastric cancer models: Augmenting its response by antiangiogenic agents [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 553.

Do dendritic cells hold the key to regulating cancer antitumor immunity?

PD-L1 blockage on dendritic cells, but not macrophages, may enhance antitumor or CD8 + T cell responses leading to greater tumor growth inhibition.

A bubble bursting-mediated oral drug delivery system that enables concurrent delivery of lipophilic and hydrophilic chemotherapeutics for treating pancreatic tumors in rats

The therapeutic outcome of pancreatic cancer remains unsatisfactory, despite many attempts to improve it. To address this challenge, an oral drug delivery system that spontaneously initiates an effervescent reaction to form gas-bubble carriers is proposed. These carriers concurrently deliver lipophilic paclitaxel (PTX) and hydrophilic gemcitabine (GEM) in the small intestine. The bursting of the bubbles promotes the intestinal absorption of the drugs. The antitumor efficacy of this proposed oral drug delivery system is evaluated in rats with experimentally created orthotopic pancreatic tumors. The combined administration of equivalent amounts of PTX and GEM via the intravenous (i.v.) route, which is clinically used for treating pancreatic cancer, serves as a control. Following oral administration, the lipophilic PTX is initially absorbed through the intestinal lymphatic system and then enters systemic circulation, whereas the hydrophilic GEM is directly taken up into the blood circulation, ultimately accumulating in the tumorous pancreatic tissues. A pharmacokinetic study reveals that the orally delivered formulation has none of the toxic side-effects that are associated with the i.v. injected formulation; changes the pharmacokinetic profiles of the drugs; and increases the bioavailability of PTX. The oral formulation has a greater impact than the i.v. formulation on tumor-specific stromal depletion, resulting in greater inhibition of tumor growth with no evidence of metastatic spread. As well as enhancing the therapeutic efficacy, this unique approach of oral chemotherapy has potential for use on outpatients, greatly improving their quality of life.

αvβ3 Integrin Antagonists Enhance Chemotherapy Response in an Orthotopic Pancreatic Cancer Model.

Pancreatic cancer decreases survival time and quality of life because of drug resistance and peripheral neuropathy during conventional treatment. This study was undertaken to investigate whether αvβ3 integrin receptor antagonist compounds NDAT and XT199 can suppress the development of cisplatin resistance and cisplatin-induced peripheral neuropathy in an orthotopic pancreatic SUIT2-luc cancer cell mouse model. Anticancer effects of these compounds and their combination with cisplatin were assessed in this tumor mouse model with bioluminescent signaling and histopathology, and a cytokine assay was used to examine expression of inflammatory cytokines IL-1β, IL-6, IL-10, and TNF-α from plasma samples. To determine the neuroprotective effects of the compounds on cisplatin-induced peripheral neuropathy, behavioral hind-limb posture of the mice was evaluated. The combination therapy of NDAT or XT199 with cisplatin elicited greater inhibition of tumor growth and increased tumor necrosis compared to cisplatin alone. NDAT and XT199 in combination with cisplatin significantly decreased expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and significantly increased expression of anti-inflammatory cytokine IL-10 in comparison to cisplatin alone. Cisplatin-treated groups showed stocking-glove hind-limb posture, whereas NDAT and XT199 with cisplatin-treated groups displayed normal hind-limb posture. Results clearly suggest that NDAT and XT199 treatment with cisplatin that inactivates NF-κB may contribute to increased antitumor and anti-inflammatory efficacy as well as alleviate cisplatin-mediated loss of motor function in this pancreatic tumor mouse model.

Possible therapeutic applicability of galectin-9 in cutaneous T-cell lymphoma

BackgroundGalectin-9, a member of the galectin family, can promote tumor growth through inducing apoptosis in anti-tumor immune cells via T cell immunoglobulin and mucin domain 3 (TIM-3). On the other hand, galectin-9 also induces tumor cell apoptosis in many malignancies and thought to have potential as an anti-cancer agent. ObjectiveTo examine the expression and therapeutic applicability of galectin-9 in cutaneous T-cell lymphoma (CTCL). MethodsGalectin-9 expression in lesional skin and sera was measured using CTCL samples. The effect of galectin-9 on CTCL cell lines was investigated in vitro. We also examined effect of galectin-9 on tumor growth of CTCL cells in immune-deficient mice. Moreover, we examined the efficacy of galectin-9, anti-TIM-3 blocking antibody, or their combination on tumor growth of EL-4 cells in wild-type mice. ResultsGalectin-9 was expressed on tumor cells in lesional skin of CTCL and the expression levels were associated with decreased CD8+ T-cell infiltration. Serum galectin-9 levels were correlated with disease severity markers. High-dose galectin-9 induced cell death of CTCL cell lines through activation of caspase-3 and caspase-9, independently of TIM-3. High-dose galectin-9 suppressed the growth of CTCL cells and EL-4 cells in vivo. Furthermore, additional anti-TIM-3 blocking antibody administration to galectin-9 achieved greater inhibition of tumor growth compared to single administration. ConclusionGalectin-9 expression on tumor cells may be associated with CTCL progression through attenuating anti-tumor immunity. On the other hand, exogenous high-dose galectin-9 administration can be a therapeutic strategy for CTCL and anti-TIM-3 blocking antibody can augment the efficacy of galectin-9.

Ultrasound-triggered breast tumor sonodynamic therapy through hematoporphyrin monomethyl ether-loaded liposome.

Sonodynamic therapy (SDT) which employs ultrasound-triggered sonosensitizers to generate reactive oxygen species (ROS) has been proved to be effective for treatment of cancers. However, it is still desirable for sonosensitizers to be delivered to tumors as effectively as possible. In this study, we prepared the hematoporphyrin monomethyl ether (HMME)-loaded liposome as the sonosensitizers for SDT and evaluated their effects on human MCF-7 breast cancer cells in vitro and in vivo. Liposomes prepared by thin film hydration technique were about 100 nm in size with positive zeta potential and exhibited spherical in shape. Following irradiation of ultrasound which generates intracellular ROS, the liposome facilitated the delivery of HMME to tumor cells. HMME-loaded liposomes showed low cytotoxicity under basal condition but significant sonodynamic effects under ultrasonic irradiation. Notably, HMME-loaded liposomes exhibited spatial distribution of HMME in tumor tissues of mice. The promoted delivery of HMME into the tumors by liposomes was shown by the greater tumor growth inhibition than free HMME after 20-day treatment. Taken together, these results show that HMME-loaded liposome functions as a promising sonosensitizer for SDT, implying the efficient antitumor effects of HMME-based SDT on breast tumor.

Abstract 3888: Intracranial evaluation of the in vivo pharmacokinetics, brain distribution, and efficacy of rucaparib in BRCA-mutant, triple-negative breast cancer

Abstract Background: The poly(ADP-ribose) polymerase inhibitor rucaparib is approved for use in recurrent ovarian cancer; however, there are limited data on the activity of rucaparib in patients with central nervous system (CNS) involvement. The goals of these studies were to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) of rucaparib and to correlate these results with the efficacy observed in a BRCA1-mutant xenograft model of triple-negative breast cancer (TNBC). In addition, a case report where rucaparib demonstrated clinical activity in the CNS of a patient with BRCA2-mutant breast cancer is presented. Methods and Results: In a PK study in CD-1 mice, rucaparib demonstrated limited brain penetration after a single oral dose of 150 mg/kg, with a mean free brain-to-plasma AUC ratio (Kpuu, brain) of 0.09. The free brain Cmax of rucaparib was 8.59 ng/mL, which was comparable to its free cerebrospinal fluid Cmax (6.69 ng/mL) but significantly lower than the total Cmax measured in the brain (118 ng/mL). The antitumor efficacy of rucaparib was evaluated in orthotopic and intracranial tumor models using the BRCA1-mutant MDA-MB-436 TNBC cell line. In the orthotopic setting, rucaparib ≥50 mg/kg BID resulted in >100% tumor growth inhibition (TGI) after 28 days of dosing. Higher levels of rucaparib were observed in the tumor relative to plasma at all doses evaluated. A PK-PD analysis showed an inverse correlation between poly(ADP-ribose) and rucaparib levels in the plasma and tumor, with decreased poly(ADP-ribose) levels correlating with greater TGI. To evaluate intracranial efficacy, a luciferase labelled MDA-MB-436 cell line was employed and tumor burden was evaluated by weekly bioluminescence measurements. Rucaparib 150 mg/kg BID demonstrated efficient suppression of tumor growth after 43 days of dosing, with >100% TGI. In support of these preclinical findings, a patient with germline BRCA2-mutant metastatic breast cancer who had progressive CNS disease following whole brain radiation therapy was treated with rucaparib 600 mg PO BID for 9 months under a compassionate use program. A reduction of multiple metastatic brain lesions was observed based on MRI scans taken before and after rucaparib treatment. The patient also experienced complete resolution of neurological symptoms. Conclusions: In vivo PK studies confirmed the limited brain penetration of rucaparib in a murine model with an intact blood-brain barrier. Nevertheless, antitumor efficacy was observed in a BRCA1-mutant intracranial TNBC mouse model, and clinical activity has been observed in a patient with germline BRCA2-mutant breast cancer and CNS involvement who was treated with rucaparib. Further studies are warranted to elucidate the activity of rucaparib in the brain. Citation Format: Minh Nguyen, Liliane Robillard, Thomas C. Harding, Jim J. Xiao, Andrew D. Simmons, Hartmut Kristeleit, Michelle (Mingxiang) Liao. Intracranial evaluation of the in vivo pharmacokinetics, brain distribution, and efficacy of rucaparib in BRCA-mutant, triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3888.

Исследование влияния ингибитора NOS Т1023 в сочетании с γ-излучением и циклофосфамидом на рост и метастазирование карциномы легких Льюис

Цель работы - изучение антинеопластических эффектов при комбинированном воздействии ингибитора NOS Т1023 с γ-излучением и циклофосфамидом. Методика. В работе использован штамм эпидермоидной карциномы легких Льюиса. Выполнено 2 независимых эксперимента: в одном изучали антинеопластические эффекты при раздельном и комбинированном воздействии Т1023 и γ-излучения, в другом - при раздельном и комбинированном воздействии Т1023 и циклофосфамида. Особям 1-й опытной группы в обоих экспериментах с 7-х по 20-е сут роста карциномы ежедневно вводили соединение Т1023 в дозе 60 мг/кг внутрибрюшинно. В первом эксперименте животные 2-й опытной группы на 7-е сут роста неоплазии получали сеанс облучения (доза 5 Гр). Во втором эксперименте животным этой группы на 7-е сут роста неоплазии вводили однократно внутрибрюшинно циклофосфамид в дозе 100 мг/кг в виде 1,0% раствора фармакопейного препарата на основе 0,9% раствора натрия хлорида. Животным 3-й опытной группы в обоих экспериментах проводили соответствующие комбинированные воздействия по этим же схемам и в таких же дозах. Первое введение Т1023 на 7-е сут роста карциномы этим животным проводили через 4 ч после облучения или введения циклофосфамида. Влияние на рост опухоли оценивали по межгрупповым различиям объемов опухолевых узлов, длительности задержки роста и индексу торможения роста опухоли. Влияние на активность метастазирования карциномы оценивали по межгрупповым различиям числа легочных метастазов на 21-е сут роста опухоли и индексу ингибирования метастазирования. Статистическую оценку значимости межгрупповых различий количественных показателей проводили с помощью дисперсионного анализа Краскела-Уоллиса с применением Q-критерия Данна. Результаты. Показано, что воздействие Т1023, как в комбинации с γ-излучением, так и в комбинации с циклофосфамидом, сопровождается статистически значимым подавлением роста и метастазирования карциномы легких Льюис. При этом антинеопластические эффекты обоих комбинированных воздействий соответствовали аддитивному противоопухолевому и антиметастатическому действию ингибитора NOS, γ-излучения и циклофосфамида. Заключение. Полученные результаты отражают способность ингибиторов NOS повышать эффективность радио- и химиотерапии злокачественных новообразований, что свидетельствуют о перспективности дальнейшей разработки препаратов типа соединения Т1023. The aim was to study antineoplastic effects of a NOS inhibitor, T1023, in combination with γ-irradiation and cyclophosphamide. Methods. Epidermoid Lewis lung carcinoma (LLC) was used as a tumor model. Two independent experiments were performed. In the first experiment, antitumor effects of T1023 and γ-irradiation were studied individually and combined. In the second experiment, antitumor effects of T1023 and cyclophosphamide were studied individually and combined. In both experiments, mice from the first experimental group were daily injected with T1023 60 mg/kg from day 7 to 20. In the first experiment, animals of the second group were treated with γ-irradiation at 5 Gy on day 7 of tumor growth. In the second experiment, mice of the second group were injected with cyclophosphamide 100 mg/kg on day 7 of tumor growth. In both experiments, mice of the third group received a respective combined treatment according to the protocols described above. The first injection of T1023 was performed on day 7 after tumor transplantation at 4 hours after the irradiation or cyclophosphamide treatment. Antitumor effects were assessed by comparing the tumor size, duration of tumor growth delay, and the index of tumor growth inhibition in control and experimental groups. The effect of treatments on metastatic activity of carcinoma was evaluated by the intergroup difference in number of pulmonary metastases inhibition index on day 21 of tumor growth. Statistical significance was determined using the Kruskal-Wallis dispersion analysis with the Dunn Q-test. Results of the study showed that the T1023 treatment both in combination with γ-irradiation and with cyclophosphamide was associated with a significantly greater inhibition of tumor growth and metastasis. The antineoplastic effect of both combinations was consistent with the additive antitumor and antimetastatic effect of the NOS inhibitor, γ-radiation, and cyclophosphamide. Conclusion. The study showed the ability of NOS inhibitors to enhance the effectiveness of radio- and chemotherapy for malignant tumors and suggested a promising outlook for further development of T1023.

PF201 THE COMBINATION OF IMGN632, A CD123-TARGETING ADC, WITH VENETOCLAX ENHANCES ANTI-LEUKEMIC ACTIVITY IN VITRO AND PROLONGS SURVIVAL IN VIVO IN PRE-CLINICAL MODELS OF HUMAN AML

Background: IMGN632 is a novel CD123-targeting antibody-drug conjugate composed of a humanized anti-CD123 antibody and a DNA-alkylating IGN payload. IMGN632 is currently in Phase I dose escalation in patients with relapsed/refractory AML or BPDCN (NCT03386513), demonstrating responses across a wide range of doses. Venetoclax, a BCL-2 inhibitor, lowers the apoptotic threshold and was recently approved for elderly AML patients in combination with azacitidine. By combining these potentially synergistic anti-leukemic mechanisms in pre-clinical AML models, the increased pro-apoptotic signaling enabled by BCL-2 inhibition would act in concert with the pro-apoptotic effects of DNA damage caused by IMGN632. Aims: The combination of IMGN632 and venetoclax was evaluated in pre-clinical AML models. Methods: The IMGN632 plus venetoclax combination was evaluated in vitro in four AML cell lines (MV4–11, MOLM-13, KG-1, EOL-1), in vivo in two subcutaneous AML xenograft models (EOL-1, KG-1), and in vivo in two AML PDX models. In subcutaneous models, tumor volume was measured, complete regressions (CRs) were noted, and endpoint was determined by either clinical observations or when tumor volume reached 1000 mm3. In PDX models, survival was measured. Results: In vitro synergy was observed in 3 of the 4 cell lines tested at doses ranging from 0.5 pM to 8 pM for IMGN632, and 0.625 nM to 20 nM for venetoclax (MV4–11, MOLM-13, EOL-1), and 1 nM to 1 μM for IMGN632, and 100 nM to 1 μM for venetoclax in the KG-1 cell line, with optimal combination indexes (CI) of 0.023 (KG-1), 0.497 (MV4–11), 0.377 (MOLM-13), and 0.67 (EOL-1), indicating strong synergy across most of the cell lines tested. In the EOL-1 model (see Figure Panel A), the combination [IMGN632 (80 μg/kg, one intravenous dose) plus venetoclax (100 mg/kg, orally, daily x28)] was highly active, resulting in 4/6 CRs and a 0% T/C (percent treatment to control of the median tumor volume, at control endpoint), compared to the corresponding inactive venetoclax (1/6 CR, 69% T/C) and active IMGN632 (2/6 CRs, 11% T/C) single agents. Importantly, the four mice treated with the combination who achieved CRs remained in remission through the end of the study at >60 days. In the IMGN632-resistant KG-1 model, the combination [IMGN632 (800 μg/kg, intravenous, weekly x3) plus venetoclax (100 mg/kg, orally, daily x28)] was active (40% T/C) while corresponding single agent IMGN632 (73% T/C) and venetoclax (81% T/C) were inactive. In a venetoclax-responsive AML PDX model (NRASm, EZH2m; see Figure Panel B), the combination [low dose IMGN632 (24 μg/kg, intravenous, weekly x3) plus venetoclax (100 mg/kg, orally, daily x28)] showed 124% ILS (% increased life span), with a median survival of 148 days. This was superior to venetoclax alone (77% ILS; median survival of 117 days) and IMGN632 alone (0% ILS; median survival of 65 days). Similarly, this same combination regimen was active (40% ILS; median survival of 108 days) in a second AML PDX model (FLT3-ITD, IDH1m), while IMGN632 (10% ILS; median survival of 85 days) and venetoclax (6% ILS; median survival of 82 days) single agents were inactive.Summary/Conclusion: The combination of IMGN632 and venetoclax demonstrated synergistic cytotoxicity in vitro and greater tumor growth inhibition and prolonged survival in vivo. Collectively, these results provide support for testing the combination of IMGN632 and venetoclax in AML patients.

Increasing Antitumor Activity of JAK Inhibitor by Simultaneous Blocking Multiple Survival Signaling Pathways in Human Ovarian Cancer

Many signaling pathways, including the JAK/STAT3 pathway, are aberrantly activated and associated with ovarian cancer growth and progression. However, inhibition of STAT3 pathway alone was not sufficient to effectively block human ovarian cancer cell survival in vitro, which could be due to the activation and compensation of multiple survival pathways. In this study, we investigated a strategy that can enhance antitumor activity of JAK/STAT3 inhibitor by combining with inhibitors targeting other growth and survival pathways. We found that the in vitro activity of JAKi was remarkably increased when additional survival pathway was blocked. Blocking SRC pathway with SRC inhibitor (SRCi) increased the efficacy of JAKi more effectively than blocking AKT or MAPK pathway. The increased activity of JAKi in combination with SRCi is synergistic and associated with attenuation of p-STAT3, p-SRC, p-AKT and p-MAPK and increased inhibition of p-AKT. Simultaneous blockade of multiple survival pathways by combining JAKi with both AKT inhibitor (AKTi) and MEK inhibitor (MEKi) also resulted in a synergistic inhibition of cell survival. Furthermore, the combined treatment of JAKi and SRCi led to an increased apoptosis and greater inhibition of tumor growth and ascites formation. Taken together, our results demonstrate that the antitumor efficacy of JAKi is improved most effectively when combined with SRCi, providing a potential combination strategy for the treatment of advanced ovarian cancer.

Combined MEK and BCL-2/XL Inhibition Is Effective in High-Grade Serous Ovarian Cancer Patient-Derived Xenograft Models and BIM Levels Are Predictive of Responsiveness.

Most patients with late-stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient-derived xenograft models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the proapoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/XL inhibitor (ABT-263) significantly reduced cell number, increased cell death, and displayed synergy in vitro in most models. In vivo, GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models-those with high BIM at baseline that was increased with MEK inhibition and those with low basal BIM and high pERK levels. Models with low BIM and low pERK were nonresponsive. Our findings demonstrate that combined MEK and BCL-2/XL inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for the clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.

Abstract P2-06-15: Rational combination of Wee1 and BCL-2 inhibition in preclinical models of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype distinguished by its lack of expression of receptors for estrogen, progesterone, and normal human epidermal growth factor 2 expression. TNBC is difficult to treat and is associated with a high risk of recurrence and mortality. In order to effectively treat TNBC, alternative therapeutic targets need to be identified. Wee1 is a tyrosine kinase that phosphorylates CDC2 to pause the cell cycle at the G2 checkpoint as a means to delay mitosis while DNA damage undergoes repair. Inactivation of Wee1 via adavosertib (AZD1775, MK1775), a highly selective inhibitor of Wee1, allows CDC25 to dephosphorylate the CDC2/cyclin B complex resulting in premature initiation of mitosis and, ultimately, mitotic catastrophe and apoptosis. An unbiased screen of adovosertib in combination with other targeted compounds in TNBC patient-derived xenograft (PDX) models demonstrated that the combination of adavosertib and navitoclax, an inhibitor of anti-apoptotic BCL-2 and BCL-XL proteins, had greater efficacy than the single agents alone. The purpose of this study was to investigate the combination of adavosertib and navitoclax in preclinical TNBC models, both in vitro and in vivo. Methods: HCC1937, CAL51, MDA-MB-231 and MDA-MB-468 cells were plated in 96-well plates and exposed to increasing concentrations of adavosertib (125nM – 1000nM), navitoclax (2500nM – 10000nM), or the combination. Cellular proliferation was assessed in real-time using IncuCyte Live Cell Analysis, followed by endpoint sulforhodamine B (SRB) assay. Combination effects were analyzed using Calcusyn to determine combination indexes (CI). Apoptosis was assessed via the Caspase 3/7 assay. Western blotting was used to assess changes in expression of CDC2, phospho-CDC2, and BCL2. TNBC PDX models CU_TNBC_013 and CU_TNBC_014 were treated with vehicle, adavosertib (50mg/kg), navitoclax (100mg/kg), or the combination and assessed for tumor growth inhibition. Results: The combination of adavosertib and navitoclax resulted in greater antiproliferative effects in vitro compared to either single agent (p< 0.05). This effect was classified as synergistic with CI values <1. We observed a significant increase in apoptosis with the combination treatment as measured by Caspase 3/7 (p <0.005). The combination of adavosertib and navitoclax treatment resulted in a decrease in phospo-CDC2, and BCL2 in cell lines. In vivo, the combination treatment resulted in greater tumor growth inhibition as compared to adavosertib or navitoclax alone in the CU_TNBC_013 and CU_TNBC_014 PDX models. Conclusions: The combination of adavosertib and navitoclax is active in preclinical TNBC models and induces apoptosis and tumor growth inhibition. This data supports the continued development of this combination in TNBC with investigation of potential selective markers. Citation Format: Simmons DM, Tse TE, Dailey K, Hartman SJ, Bagsby S, Pitts TM, Tentler JJ, Diamond JR. Rational combination of Wee1 and BCL-2 inhibition in preclinical models of triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-15.

Abstract A187: RIG-I agonists reinforce antitumor adaptive immunity and decrease Treg activity in breast cancer

Abstract RIG-I like receptors, RNA helicases that sense viral oligonucleotide motifs and activate innate immunity, are gaining interest in cancer therapy, given their ability to redirect immune responses within the tumor microenvironment (TME), and increase efficacy of experimental cancer vaccines. RIG-I agonists are not well studied in breast cancers, a type of cancer that is often considered immunologically “silent.” We recently reported that therapeutic delivery of RIG-I agonists increase tumor-infiltrating leukocytes (TILs) and expression of proinflammatory Th1 cytokines in the 4T1 mouse model of aggressive, metastatic breast cancer through tumor cell-intrinsic mechanisms. However, these studies do not rule out the importance of myeloid immune responders (e.g., macrophages and dendritic cells) in propagating the effects of RIG-I agonists against tumor cells in vivo, nor do they rule out the impact of RIG-I agonists on adaptive antitumor immunity, a subject that is relatively understudied. We assessed the effects of the RIG-I agonist SLR20 on the the activity of effector T-lymphocytes (TEff) and regulatory T-lymphocytes (TReg) in the TME. Interestingly, SLR20 treatment of mouse and human breast tumor cells increased expression of FAS and MHC-I on tumor cells, and caused tumor cells to express T-cell chemoattractants (e.g., CXCL10, RANTES), potentially increasing T-cells recruitment to tumors, and increasing tumor cell susceptibility to TEff recognition and killing. Using an ex vivo co-culture assay in which 4T1 mouse mammary tumor cells were co-cultured with CD8+ T-cells harvested from mice pre-inoculated with SLR20-treated 4T1 tumor cells, we measured the rate of CD8+-mediated tumor cell killing. This approach revealed that T-cells harvested from mice inoculated with SLR20-treated cells caused greater tumor cell killing than what was seen by CD8+ T-cells harvested from untreated mice. We also found that conditioned media harvested from 4T1 cells treated with SLR20 increased clonal expansion of CD3/CD28-activated T-cells above what was seen with conditioned media harvested from 4T1 cells treated with a control oligonucleotide or from untreated 4T1 cells. TGFβ-mediated differentiation of CD4+ T-cells into tolerogenic and immunosuppressive TRegs was measured in cultures of CD4+ T-cells treated with cultured media derived from SLR20-treated 4T1 cells. These studies showed that cultured media harvested from 4T1 cells treated with SLR20, but not from untreated 4T1 cells or 4T1 cells treated with a control ligand, diminished TReg differentiation, and decreased CD4+ T-cells surface expression of PD-1, CTLA4, and CCR8. Importantly, in vivo experiments assessing therapeutic treatment of 4T1 tumors with SLR20 revealed greater tumor growth inhibition when SLR20 was combined with PD-L1 targeting antibodies. Taken together, these findings indicate that therapeutic activation of RIG-I signaling operates at the interface of innate and adaptive immunity within breast tumors to redirect the TME from an immunosuppressed state to one that is immunogenic and receptive to clinically relevant checkpoint inhibitors. Citation Format: David L. Elion, Max E. Jacobson, Donna J. Hicks, Bushra Rahman, Violeta Sanchez, Paula I Gonzales-Ericsson, Olga Fedorova, Anna M. Pyle, John T. Wilson, Rebecca S. Cook. RIG-I agonists reinforce antitumor adaptive immunity and decrease Treg activity in breast cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A187.

Role of FcγRs in Antibody-Based Cancer Therapy.

Monoclonal antibodies can mediate antitumor activity by multiple mechanisms. They can bind directly to tumor receptors resulting in tumor cell death, or can bind to soluble growth factors, angiogenic factors, or their cognate receptors blocking signals required for tumor cell growth or survival. Monoclonal antibodies, upon binding to tumor cell, can also engage the host's immune system to mediate immune-mediated destruction of the tumor. The Fc portion of the antibody is essential in engaging the host immune system by fixing complement resulting in complement-mediated cytotoxicity (CDC) of the tumor, or by engaging Fc receptors for IgG (FcγR) expressed by leukocytes leading to antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Antibodies whose Fc portion preferentially engage activating FcγRs have shown greater inhibition of tumor growth and metastasis. Monoclonal antibodies can also stimulate the immune system by binding to targets expressed on immune cells. These antibodies may stimulate antitumor immunity by antagonizing a negative regulatory signal, agonizing a costimulatory signal, or depleting immune cells that are inhibitory. The importance of Fc:FcγR interactions in antitumor therapy for each of these mechanisms have been demonstrated in both mouse models and clinical trials and will be the focus of this chapter.

Synergistic Antimyeloma Activity of Dendritic Cells and Pomalidomide in a Murine Myeloma Model.

We have previously shown that immunization with tumor antigen-loaded dendritic cells (DCs) and the immunomodulating drug, lenalidomide, synergistically potentiates the enhancing antitumor immunity in a myeloma mouse model. In this study, we investigated the immunogenicity of DCs combined with pomalidomide and dexamethasone in a myeloma mouse model. MOPC-315 cells were injected subcutaneously to establish myeloma-bearing mice. Four test groups were used to mimic clinical protocol: (1) PBS control, (2) DCs, (3) pomalidomide + dexamethasone, and (4) DCs + pomalidomide + dexamethasone. The combination of DCs plus pomalidomide and dexamethasone displayed greater inhibition of tumor growth compared to the other groups. This effect was closely related with reduced numbers of immune suppressor cells including myeloid-derived suppressor cells, M2 macrophages, and regulatory T cells, with the induction of immune effector cells such as CD4+ and CD8+ T cells, memory T cells, natural killer (NK) cells, and M1 macrophages, and with the activation of T lymphocytes and NK cells in the spleen. Moreover, the level of the immunosuppressive factor vascular endothelial growth factor was significantly reduced in the tumor microenvironment. The collective findings in the murine myeloma model suggest that tumor antigen-loaded DCs combined with pomalidomide and dexamethasone synergistically enhance antitumor immunity by skewing the immune-suppressive status toward an immune-supportive status.

STK405759 as a combination therapy with bortezomib or dexamethasone, in in vitro and in vivo multiple myeloma models.

Multiple myeloma (MM) remains an incurable hematological malignancy. Combination regimens of conventional and novel drugs have improved patient’s survival. However, most patients inevitably relapse and become refractory to the current therapeutic armamentarium.We investigated the efficacy of combining the microtubule-targeting agent STK405759 with dexamethasone or bortezomib in vitro and in vivo.STK405759 combined with dexamethasone or bortezomib had synergistic cytotoxic activity in RPMIS, CAG and MM1.S human MM cell lines through activation of caspase 2, 3, 8, 9 and PARP. These treatments remained cytotoxic in the presence of bone marrow stroma cells. In other MM cells, including cells resistant to vincristine, melphalan, mitoxantrone or dexamethasone, these combinations decreased significantly survival as compared to single agents.In in vivo studies, STK405759 disrupted existing blood vessels in xenograft tumors, acting not only as a cytotoxic agent but also as an anti-angiogenic drug. Mice treated with STK405759 in combination with dexamethasone or bortezomib resulted in greater tumor growth inhibition, increased overall response and prolonged survival as compared to as compared to BTZ or DEXA alone. Their anticancer activity was mediated by activation of apoptosis and reduction of tumor microvessel density.These preclinical studies provide the rationale for future clinical trials of STK405759, dexamethasone and bortezomib combinations to improve the outcome of multiple myeloma patients.

Abstract 4829: Plumbagin amplifies the pro-apoptotic and anti-tumorigenic activities of vemurafenib in BRAF-mutant melanoma

Abstract Melanoma, the deadliest form of skin cancer, originates from the pigment-producing cells in the skin known as melanocytes. The incidence of melanoma is increasing at a more alarming rate than any other cancer worldwide. BRAF mutations occur in ~50% of melanomas that drive oncogenic activation of the BRAF-MEK-ERK resulting in cell proliferation and tumorigenesis. BRAF and MEK inhibitors either alone or in combination have improved the overall survival of melanoma patients with BRAF mutations. However, these patients inevitably develop resistance to these treatments. Thus, there is a need for exploring novel agents to enhance the therapeutic efficacy of these inhibitors for the management of melanoma. Phytochemicals offer promising new options for the development of more effective strategies because of their relatively low toxicities on normal cells. Plumbagin, a naturally occurring naphthoquinone found in flowers and roots of the Plumbaginaceae family, exhibits potent anti-proliferative and anti-tumorigenic properties. Recently, we have shown that plumbagin reduces proliferation and induces apoptosis in melanoma cells by downregulating PI3K signaling. Studies have unequivocally demonstrated that BRAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways play major roles in melanoma development and therapy resistance. Therefore, the objective of this study is to determine whether plumbagin in combination with vemurafenib could offer therapeutic benefit over an individual agent for the treatment of the BRAF-mutant melanoma. Treatment of BRAF-mutant human melanoma cells with plumbagin and vemurafenib in combination more effectively reduced cell growth and colony formation than individual agents. Combination treatment (plumbagin + vemurafenib) also resulted in increased apoptosis as revealed by cleaved caspase-3 and PARP. Furthermore, combination treatment resulted in enhanced expression of Bax and Bak, and increased inhibition of Bcl2 and Mcl-1. We also found that combination treatment was more effective in inhibiting the phosphorylation of MEK1/2 ERK1/2, AKT, and mTOR as well as the protein expression of PI3K. In athymic nude mice subcutaneously implanted with A375 melanoma cells, combination treatment resulted in greater inhibition of tumor growth, when compared to individual agents. Combination treatment also resulted in (i) decreased phosphorylation of MEK1/2, ERK1/2, AKT, and mTOR (ii) decreased proliferation, and (iii) increased apoptosis in tumor samples than individual agents.These data encourage the need for further advanced in vivo studies to evaluate the effects of plumbagin as an adjuvant to current therapies for the management of BRAF-mutant melanoma. Citation Format: Pooja Sharma, Mary Katherine Montes de Oca, Ross L. Pearlman, Farrukh Afaq. Plumbagin amplifies the pro-apoptotic and anti-tumorigenic activities of vemurafenib in BRAF-mutant melanoma [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 4829.

Abstract 4948: MDM2 antagonism overcomes resistance to CDK4/6 inhibition in melanoma

Abstract Metastatic melanoma is an aggressive disease characterized by resistance to conventional cancer treatments such as radiation and chemotherapy. While immune checkpoint blockade and BRAF inhibitor therapies are effective in many melanoma cases, a significant proportion of patients are resistant to these standard of care treatments and thus require new therapeutic approaches. We hypothesized that targeting CDK4/6 may offer hope to melanoma patients refractory to immunotherapy and BRAF inhibition, based on CDK4 being a key oncogene activated by mutations or deletions of CDKN2A gene, which are very common in melanoma (>50% of cases). However, in vivo studies using our extensive collection of melanoma patient-derived xenografts (PDX) showed that the majority of melanoma tumors are intrinsically resistant to CDK4/6 inhibition. We identified the mechanism of this resistance which was driven by cooperative activities of other CDKs expressed by tumor cells that compensated for the loss of CDK4/6 function to allow cell cycle progression through G1 and S phases. In order to overcome compensatory activation of other CDKs, we used small molecule antagonists of MDM2-p53 interaction, that stabilize p53 protein, which, in turn, upregulates an endogenous pan-CDK inhibitor, p21. Notably, combined MDM2 and CDK4/6 antagonism resulted in greater inhibition of tumor growth than either inhibitor individually. This synergistic effect was observed in multiple melanoma PDX models and in immunocompetent mice bearing mouse melanoma tumors. In summary, our preclinical data suggest that combining CDK4/6 inhibitors with MDM2 antagonists is a promising strategy for the treatment of metastatic melanoma, which should be further tested by a clinical study. Citation Format: Anna E. Vilgelm, Nabil Saleh, Kelsie Riemenschneider, Lauren Slesur, Sheau-Chian Chen, Gregory D. Ayers, Rondi M. Kauffman, Mark C. Kelley, Douglas B. Johnson, Ann Richmond. MDM2 antagonism overcomes resistance to CDK4/6 inhibition in melanoma [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 4948.