macrophages mediating radioresistance of IBC cells in vitro, in part, through upregulation of PKCz. Herein, we examined the role of PKCz on intrinsic radiation resistance in IBC cell lines. Materials/Methods: PRKCZ expression was examined in the TCGA breast cancer data using UALCAN. IBC cells comprising various intrinsic subtypes of breast cancer (Her2 positive IBC3, KPL4, and SUM190; and Triple negative breast cancer cell line SUM149) were transfected with CRISPR/Cas9 targeting PKCz. PKCz knockout (KO) clones were validated using immunoblotting with anti-PKCz (cell signaling technology #9372). 2D clonogenic assays were performed as described. Results: PRKCZ expression was significantly higher in tumor vs normal breast cancer (P < 0.0001) in all subtypes. Expression was similar across stage, age, and subtype although lower in HER2+ tumors. Expression in the world consortium IBC data N Z 389 breast cancers, (IBC Z 137; and nIBC Z 252) revealed no difference in IBC vs non-IBC. Baseline expression of PKCz was detected at least 100-fold higher in IBC3 (136 fold), SUM190 (128 fold), and SUM149 (109 fold) cells, relative to KPL4 cells. In vitro 2D clonogenic assays demonstrate a significant radiosensitizing effect in SUM149 IBC cells with PKCz knockdown (SF2 Z 0.3), relative to parental (SF2 Z 0.4) and cells transfected with control CRISPR plasmids (SF2 Z 0.46). Conclusion: Our preliminary data suggest PKCz as an intrinsic mediator of radioresistance of IBC cells independent of the previously described microenvironment response effect. In vivo whole brain radiosensitization studies with IBC cells and the immune competent 4T1 mouse model are planned.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Thirty percent of patients will experience locoregional recurrence for which median survival is less than 1 year. Factors contributing to treatment failure include inherent resistance to X-rays and chemotherapy, hypoxia, epithelial to mesenchymal transition, and immune suppression. The unique properties of 12C radiotherapy including enhanced cell killing, a decreased oxygen enhancement ratio, generation of complex DNA damage, and the potential to overcome immune suppression make its application well suited to the treatment of HNSCC. We examined the 12C radioresponse of five HNSCC cell lines, whose surviving fraction at 3.5 Gy ranged from average to resistant when compared with a larger panel of 38 cell lines to determine if 12C irradiation can overcome X-ray radioresistance and to identify biomarkers predictive of 12C radioresponse. Cells were irradiated with 12C using a SOBP with an average LET of 80 keV/μm (CNAO: Pavia, Italy). RBE values varied depending upon endpoint used. A 37 gene signature was able to place cells in their respective radiosensitivity cohort with an accuracy of 86%. Radioresistant cells were characterized by an enrichment of genes associated with radioresistance and survival mechanisms including but not limited to G2/M Checkpoint MTORC1, HIF1α, and PI3K/AKT/MTOR signaling. These data were used in conjunction with an in silico-based modeling approach to evaluate tumor control probability after 12C irradiation that compared clinically used treatment schedules with fixed RBE values vs. the RBEs determined for each cell line. Based on the above analysis, we present the framework of a strategy to utilize biological markers to predict which HNSCC patients would benefit the most from 12C radiotherapy.
Stereotactic Ablative Radiotherapy (SAbR) has revolutionized the treatment of non-small cell lung cancer (NSCLC). Despite these advances, treatment-limiting normal tissue toxicities preclude the use of fully potent radiation prescriptions in large or centrally located tumors. Radiation-induced lung fibrosis (RILF), thought to be generated by the production of superoxide in irradiated tissues, is a natural target for agents to limit RILF and thus allow for fully potent SAbR. Superoxide dismutase (SOD) catalyzes the conversion of superoxide into hydrogen peroxide, which then may be converted to water and oxygen via catalase, protecting normal cells from oxidative damage. GC4419 (Galera Therapeutics, St. Louis, MO), a selective small molecule SOD-mimetic acts as both a protector and mitigator of radiation-induced superoxide damage. A single pretreatment with GC4419 significantly reduced the fibrotic density of focally irradiated murine lung tissue (54 Gy, single dose). Furthermore, daily post-irradiation (54 Gy, single dose) use of GC4419 also increasingly reduced lung fibrosis based upon the total duration of daily delivery. Since tumor protection could be a concern, animals with H1299, A549, and HCC827 lung tumor xenografts were treated with GC4419 30 minutes prior to the tumors being irradiated with a single 18 Gy dose, followed by 4 additional daily doses of GC4419. Tumor growth was significantly delayed (p = 0.0022) with the majority of mice apparently tumor-free at X days. Similar enhancements in tumor radiation response were seen with syngeneic lung (LLC) and breast (4T1) tumor models. Subsequent Tumor Cure Dose (TCD50) assays demonstrated that GC4419 enhanced the efficacy of radiation by a factor of 1.67. The dose enhancement seen with GC4419 was associated with the size of the dose per fraction, and if the single 18 Gy dose fractionation scheme was altered to include the biologically equivalent dose schedules of daily irradiations of 2 Gy for 16 days, 4.8 Gy for 5 days, 7.3 Gy for 3 days, or 9.9 Gy for 2 days, the radiation enhancing properties of GC4419 were more pronounced as the dose per fraction increased. Our hypothesis that the tumor response was driven by the overwhelming and persistent production of H2O2 is supported by the fact that the GC4419-enhanced radiation response of tumors derived from H1299CAT cells (doxycycline-driven catalase overexpressing), and treated accordingly with doxycycline, is completely abrogated. These findings suggest that GC4419 not only displays clinical potential as a normal tissue radiation protector and mitigator, but has the additional advantage of enhancing SAbR. Citation Format: Brock J. Sishc, Elizabeth Polsdofer, David A. Bloom, Collin Heer, Douglas R. Spitz, Debabrata Saha, Michael D. Story. The radioprotector GC4419 ameliorates radiation induced lung fibrosis while enhancing the response of non-small cell lung cancer tumors to high dose per fraction radiation exposures [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 667.
Locoregional recurrence (LRR) is the major cause of morbidity and mortality in patients with squamous cell carcinoma of the head and neck (HNSCC). For those patients that experience surgically unresectable LRR, median overall survival (OS) is less than one year. Approaches utilizing stereotactic ablative radiotherapy (SAbR) and immune oncology agents such as Nivolumab (α-PD-1 agonist) in the salvage setting have become alternatives to conventional chemoradiation therapy. Despite improvements in OS, rates of acute oral mucositis (OM) and late toxicities (skin fibrosis) remain a concern with SAbR, particularly in the context of re-irradiation. Here we present pre-clinical evidence that GC4419 (Galera Therapeutics), a highly selective superoxide dismutase mimetic that recently received FDA Breakthrough therapy status following a Phase II, randomized, placebo controlled trial for reducing the duration and incidence of severe OM in patients undergoing chemoradiation therapy for locally advanced HNSCC (NCT02508389) also enhances the anti-tumor radiation response of HNSCC tumors to irradiation. We demonstrate in pre-clinical animal models that GC4419 protects the normal mucosa of the mouse tongue from radiation exposure. Utilizing clonogenic survival assays, GC4419 demonstrates single agent anti-cancer activity at physiologically achievable concentrations, while also enhancing the response of HNSCC lines to irradiation. In tumor growth delay (TGD) experiments utilizing the syngeneic HNSCC tumor model, AT-84, when GC4419 is delivered 30-60 minutes pre-irradiation with biologically equivalent fractionation schedules of 17 Gy x 1 fxn, 10.24 Gy x 2 fxn, or 5 Gy x 5 fxn, an enhancement of the radiation response is achieved. In tumor cure rate (TCD50) studies, GC4419 enhanced radiation exposure with a DEF of 1.25. Furthermore, when GC4419 is combined with radiation and an α-PD-1 inhibitor, a further enhancement of the radiation response is observed, indicating that GC4419 compliments radiotherapy combined with immune oncology. We will also report the results of ongoing studies examining the potency of GC4419 in protecting normal murine mucosa in the setting of reirradiation relevant to SAbR as a salvage therapy. This potentiation of the anti-tumor response with GC4419 is more pronounced at doses exceeding the threshold to be considered for intensity modulated radiation therapy (IMRT). The accompanying pre-clinical data strongly suggest that GC4419 should be combined with radio-immune therapy to not only enhance local tumor control, but that the potential for normal tissue protection with SAbR also creates the opportunity for dose escalation and may further improvement in treatment outcome. Citation Format: Brock James Sishc, Elizabeth Polsdofer, Debabrata Saha, Michael Story. GC4419 protects again radiation induced oral mucositis, enhances the response of squamous cell carcinoma of the head and neck tumors to ionizing radiation, and enhances radioimmune therapy [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 6284.
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