AZD8055 enhances in vivo efficacy of afatinib in chordomas.

Chordomas are primary bone tumors that arise in the cranial base, mobile spine, and sacrococcygeal region, affecting patients of all ages. Currently, there are no approved agents for chordoma patients. Here, we evaluated the anti-tumor efficacy of small molecule inhibitors that target oncogenic pathways in chordoma, as single agents and in combination, to identify novel therapeutic approaches with the greatest translational potential. A panel of small molecule compounds was screened in vivo against patient-derived xenograft (PDX) models of chordoma, and potentially synergistic combinations were further evaluated using chordoma cell lines and xenograft models. Among the tested agents, inhibitors of EGFR (BIBX 1382, erlotinib, and afatinib), c-MET (crizotinib), and mTOR (AZD8055) significantly inhibited tumor growth in vivo but did not induce tumor regression. Co-inhibition of EGFR and c-MET using erlotinib and crizotinib synergistically reduced cell viability in chordoma cell lines but did not result in enhanced in vivo activity. Co-inhibition of EGFR and mTOR pathways using afatinib and AZD8055 synergistically reduced cell viability in chordoma cell lines. Importantly, this dual inhibition completely suppressed tumor growth in vivo, showing improved tumor control. Together, these data demonstrate that individual inhibitors of EGFR, c-MET, and mTOR pathways suppress chordoma growth both in vitro and in vivo. mTOR inhibition increased the efficacy of EGFR inhibition on chordoma growth in several preclinical models. The insights gained from our study potentially provide a novel combination therapeutic strategy for patients with chordoma. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

TNIK inhibition sensitizes TNIK-overexpressing lung squamous cell carcinoma to radiotherapy.

Most patients with lung squamous cell carcinoma (LSCC) undergo chemotherapy, radiotherapy, and adjuvant immunotherapy for locally advanced disease. The efficacy of these treatments is still limited due to dose-limiting toxicity or locoregional recurrence. New combination approaches and targets such as actionable oncogenic drivers are needed to advance treatment options for LSCC patients. Moreover, other options for chemotherapy-ineligible patients are also limited. As such there is a critical need for the development of selective and potent chemoradiosensitizers for locally advanced LSCC. Here, we investigated inhibiting TRAF2 and NCK-interacting protein kinase (TNIK), which is amplified in 40% of LSCC patients, as a strategy to sensitize LSCC tumors to chemo- and radiotherapy. Employing a range of human LSCC cell lines and the TNIK inhibitor NCB-0846, we investigated the potential of TNIK as a chemo- and radiosensitizing target with in vitro and in vivo preclinical models. The combination of NCB-0846 with cisplatin or etoposide was at best additive. Interestingly, pre-treating LSCC cells with NCB-0846 prior to ionizing radiation (IR) potentiated the cytotoxicity of IR in a TNIK-specific fashion. Characterization of the radiosensitization mechanism suggested that TNIK inhibition may impair the DNA damage response and promote mitotic catastrophe in irradiated cells. In a subcutaneous xenograft in vivo model, pretreatment with NCB-0846 significantly enhanced the efficacy of IR and caused elevated necrosis in TNIKhigh LK2 tumors but not TNIKlow KNS62 tumors. Overall, these results indicate that TNIK inhibition may be a promising strategy to increase the efficacy of radiotherapy in LSCC patients with high TNIK expression.

TNIK Inhibition Sensitizes TNIK-Overexpressing Lung Squamous Cell Carcinoma to Radiotherapy.

Most patients with lung squamous cell carcinoma (LSCC) undergo chemotherapy, radiotherapy, and adjuvant immunotherapy for locally advanced disease. The efficacy of these treatments is still limited because of dose-limiting toxicity or locoregional recurrence. New combination approaches and targets such as actionable oncogenic drivers are needed to advance treatment options for patients with LSCC. Moreover, other options for chemotherapy-ineligible patients are limited. As such, there is a critical need for the development of selective and potent chemoradiosensitizers for locally advanced LSCC. In this study, we investigated inhibiting TRAF2- and NCK-interacting protein kinase (TNIK), which is amplified in 40% of patients with LSCC, as a strategy to sensitize LSCC tumors to chemotherapy and radiotherapy. Employing a range of human LSCC cell lines and the TNIK inhibitor NCB-0846, we investigated the potential of TNIK as a chemo- and radiosensitizing target with in vitro and in vivo preclinical models. The combination of NCB-0846 with cisplatin or etoposide was at best additive. Interestingly, pre-treating LSCC cells with NCB-0846 prior to ionizing radiation (IR) potentiated the cytotoxicity of IR in a TNIK-specific fashion. Characterization of the radiosensitization mechanism suggested that TNIK inhibition may impair the DNA damage response and promote mitotic catastrophe in irradiated cells. In a subcutaneous xenograft in vivo model, pretreatment with NCB-0846 significantly enhanced the efficacy of IR and caused elevated necrosis in TNIKhigh LK2 tumors but not TNIKlow KNS62 tumors. Overall, these results indicate that TNIK inhibition may be a promising strategy to increase the efficacy of radiotherapy in patients with LSCC with high TNIK expression.

Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice.

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.

Griseofulvin Radiosensitizes Non-Small Cell Lung Cancer Cells and Activates cGAS.

Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non-small cell lung cancer (NSCLC). Griseofulvin (GF; FDA-approved treatment) inhibits CC, and combined with radiation treatment (RT), resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability in vitro. In vivo, GF treatment was well tolerated by mice, and the combined therapy of GF and radiation treatment resulted in a significant tumor growth delay. Both GF and radiation treatment also induced the generation of micronuclei (MN) in vitro and in vivo and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased radiation treatment efficacy in lung cancer preclinical models in vitro and in vivo. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.

Nelfinavir Inhibits the Growth of Small-cell Lung Cancer Cells and Patient-derived Xenograft Tumors.

BACKGROUND/AIM: Small-cell lung cancer (SCLC) is aggressive and confers poor prognosis. Although SCLC shows more response to chemotherapy than other types of lung cancer, it is difficult to cure because of its frequent recurrence. New drugs and molecular targets need to be identified. MATERIALS AND METHODS: We investigated the effect of nelfinavir, an HIV protease inhibitor, on SCLC cells and in preclinical treatment studies using SCLC patient-derived xenograft (PDX) mouse models. RESULTS: Nelfinavir inhibited SCLC cell proliferation and induced cell death in vitro, which was caused by induction of the unfolded protein response (UPR), inhibition of mammalian/mechanistic target of rapamycin (mTOR) activation, and reduction in the expression of SCLC-related molecules such as achaete-scute homolog 1 (ASCL1). In vivo, nelfinavir inhibited the growth of SCLC PDX tumors, which correlated with the induction of UPR and reduced expression of ASCL1. CONCLUSION: Nelfinavir is highly effective in SCLC in vitro and in vivo, suggesting possible incorporation of nelfinavir into clinical trials for patients with SCLC.

Regulation of RhoB Gene Expression during Tumorigenesis and Aging Process and Its Potential Applications in These Processes.

RhoB, a member of the Ras homolog gene family and GTPase, regulates intracellular signaling pathways by interfacing with epidermal growth factor receptor (EGFR), Ras, and phosphatidylinositol 3-kinase (PI3K)/Akt to modulate responses in cellular structure and function. Notably, the EGFR, Ras, and PI3K/Akt pathways can lead to downregulation of RhoB, while simultaneously being associated with an increased propensity for tumorigenesis. Functionally, RhoB, part of the Rho GTPase family, regulates intracellular signaling pathways by interfacing with EGFR, RAS, and PI3K/Akt/mammalian target of rapamycin (mTOR), and MYC pathways to modulate responses in cellular structure and function. Notably, the EGFR, Ras, and PI3K/Akt pathways can lead to downregulation of RhoB, while simultaneously being associated with an increased propensity for tumorigenesis. RHOB expression has a complex regulatory backdrop consisting of multiple histone deacetyltransferase (HDACs 1 and 6) and microRNA (miR-19a, -21, and -223)-mediated mechanisms of modifying expression. The interwoven nature of RhoB's regulatory impact and cellular roles in regulating intracellular vesicle trafficking, cell motion, and the cell cycle lays the foundation for analyzing the link between loss of RhoB and tumorigenesis within the context of age-related decline in RhoB. RhoB appears to play a tissue-specific role in tumorigenesis, as such, uncovering and appreciating the potential for restoration of RHOB expression as a mechanism for cancer prevention or therapeutics serves as a practical application. An in-depth assessment of RhoB will serve as a springboard for investigating and characterizing this key component of numerous intracellular messaging and regulatory pathways that may hold the connection between aging and tumorigenesis.

Bisphosphonamidate clodronate prodrug exhibits selective cytotoxic activity against melanoma cell lines.

Bisphosphonates are used clinically to treat disorders of calcium metabolism and malignant bone disease and are known to inhibit cancer cell growth, adhesion, and invasion. However, clinical use of these agents for the treatment of extraskeletal disease is limited because of low cell permeability. We recently described a bisphosphonamidate prodrug strategy for efficient intracellular release of bisphosphonates, including clodronate (CLO), in non-small cell lung cancer cells. To evaluate anticancer activity of this prodrug class across many cancer cell types, the bisphosphonamidate clodronate prodrug (CLO prodrug) was screened against the NCI-60 cell line panel, and was found to exhibit selectivity toward melanoma cell lines. Here, we confirm efficient cellular uptake and intracellular activation of this prodrug class in melanoma cells. We further demonstrate inhibition of melanoma cell proliferation, induction of apoptosis, and an antitumor effect of CLO prodrug in a xenograft model. These data suggest a novel therapeutic application for the CLO prodrug and potential to selectively target melanoma cells.

Rapamycin rescues ABT-737 efficacy in small cell lung cancer.

Overexpression of the antiapoptotic protein Bcl-2 is observed in the majority of small cell lung cancer (SCLC) cases and is associated with resistance to chemotherapy. While targeting Bcl-2 in hematologic malignancies continues to show signs of promise, translating the BH3 mimetic ABT-737 (or ABT-263; navitoclax) to the clinic for solid tumors has remained problematic, with limited single-agent activity in early-phase clinical trials. Here, we used patient-derived xenograft (PDX) models of SCLC to study ABT-737 resistance and demonstrated that responses to ABT-737 are short lived and coincide with decreases in HIF-1α-regulated transcripts. Combining the mTOR inhibitor rapamycin with ABT-737 rescued this resistance mechanism, was highly synergistic in vitro, and provided durable tumor regressions in vivo without notable hematologic suppression. In comparison, tumor regressions did not occur when ABT-737 was combined with etoposide, a gold-standard cytotoxic for SCLC therapy. Rapamycin exposure was consistently associated with an increase in the proapoptotic protein BAX, whereas ABT-737 caused dose-dependent decreases in BAX. As ABT-737 triggers programmed cell death in a BAX/BAK-dependent manner, we provide preclinical evidence that the efficacy of ABT-737 as a single agent is self-limiting in SCLC, but the addition of rapamycin can maintain or increase levels of BAX protein and markedly enhance the anticancer efficacy of ABT-737. These data have direct translational implications for SCLC clinical trials.

Poly(ß-amino ester) nanoparticle delivery of TP53 has activity against small cell lung cancer in vitro and in vivo.

Small cell lung cancer (SCLC) is an aggressive disease with one of the highest case-fatality rates among cancer. The recommended therapy for SCLCs has not changed significantly over the past 30 years; new therapeutic approaches are a critical need. TP53 is mutated in the majority of SCLC cases and its loss is required in transgenic mouse models of the disease. We synthesized an array of biodegradable poly(ß-amino ester) (PBAE) polymers that self-assemble with DNA and assayed for transfection efficiency in the p53-mutant H446 SCLC cell line using high-throughput methodologies. Two of the top candidates were selected for further characterization and TP53 delivery in vitro and in vivo. Nanoparticle delivery of TP53 resulted in expression of exogenous p53, induction of p21, induction of apoptosis, and accumulation of cells in sub-G1 consistent with functional p53 activity. Intratumoral injection of subcutaneous H446 xenografts with polymers carrying TP53 caused marked tumor growth inhibition. This is the first demonstration of TP53 gene therapy in SCLC using nonviral polymeric nanoparticles. This technology may have general applicability as a novel anticancer strategy based on restoration of tumor suppressor gene function.

Erlotinib inhibits growth of a patient-derived chordoma xenograft.

Chordomas are rare primary bone tumors that occur along the neuraxis. Primary treatment is surgery, often followed by radiotherapy. Treatment options for patients with recurrence are limited and, notably, there are no FDA approved therapeutic agents. Development of therapeutic options has been limited by the paucity of preclinical model systems. We have established and previously reported the initial characterization of the first patient-derived chordoma xenograft model. In this study, we further characterize this model and demonstrate that it continues to resemble the original patient tumor histologically and immunohistochemically, maintains nuclear expression of brachyury, and is highly concordant with the original patient tumor by whole genome genotyping. Pathway analysis of this xenograft demonstrates activation of epidermal growth factor receptor (EGFR). In vitro studies demonstrate that two small molecule inhibitors of EGFR, erlotinib and gefitinib, inhibit proliferation of the chordoma cell line U-CH 1. We further demonstrate that erlotinib significantly inhibits chordoma growth in vivo. Evaluation of tumors post-treatment reveals that erlotinib reduces phosphorylation of EGFR. This is the first demonstration of antitumor activity in a patient-derived chordoma xenograft model and these findings support further evaluation of EGFR inhibitors in this disease.

Characterizing the behavioral effects of nerve agent-induced seizure activity in rats: increased startle reactivity and perseverative behavior.

The development and deployment of next-generation therapeutics to protect military and civilian personnel against chemical warfare nerve agent threats require the establishment and validation of animal models. The purpose of the present investigation was to characterize the behavioral consequences of soman (GD)-induced seizure activity using a series of behavioral assessments. Male Sprague-Dawley rats (n=24), implanted with a transmitter for telemetric recording of encephalographic signals, were administered either saline or 1.0 LD50 GD (110 µg/kg, sc) followed by treatment with a combination of atropine sulfate (2 mg/kg, im) and the oxime HI-6 (93.6 mg/kg, im) at 1 min post-exposure. Seizure activity was allowed to continue for 30 min before administration of the anticonvulsant diazepam (10 mg/kg, sc). The animals that received GD and experienced seizure activity had elevated startle responses to both 100- and 120-dB startle stimuli compared to control animals. The GD-exposed animals that had seizure activity also exhibited diminished prepulse inhibition in response to 120-dB startle stimuli, indicating altered sensorimotor gating. The animals were subsequently evaluated for the acquisition of lever pressing using an autoshaping procedure. Animals that experienced seizure activity engaged in more goal-directed (i.e., head entries into the food trough) behavior than did control animals. There were, however, no differences between groups in the number of lever presses made during 15 sessions of autoshaping. Finally, the animals were evaluated for the development of fixed-ratio (FR) schedule performance. Animals that experienced GD-induced seizure activity engaged in perseverative food trough-directed behaviors. There were few differences between groups on other measures of FR schedule-controlled behavior. It is concluded that the GD-induced seizure activity increased startle reactivity and engendered perseverative responding and that these measures are useful for assessing the long-term effects of GD exposure in rats.