Lauryn R. Werner

In situ tumor vaccination by combining local radiation and tumor-specific antibody or immunocytokine treatments

Interest in combining radiotherapy and immune checkpoint therapy is growing rapidly. In this study, we explored a novel combination of this type to augment antitumor immune responses in preclinical murine models of melanoma, neuroblastoma, and head and neck squamous cell carcinoma. Cooperative effects were observed with local radiotherapy and intratumoral injection of tumor-specific antibodies, arising in part from enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). We could improve this response by combining radiation with intratumoral injection of an IL2-linked tumor-specific antibody (termed here an immunocytokine), resulting in complete regression of established tumors in most animals associated with a tumor-specific memory T-cell response. Given the T-cell response elicited by combined local radiation and intratumoral immunocytokine, we tested the potential benefit of adding this treatment to immune checkpoint blockade. In mice bearing large primary tumors or disseminated metastases, the triple-combination of intratumoral immunocytokine, radiation, and systemic anti-CTLA-4 improved primary tumor response and animal survival compared with combinations of any two of these three interventions. Taken together, our results show how combining radiation and intratumoral immunocytokine in murine tumor models can eradicate large tumors and metastases, eliciting an in situ vaccination effect that can be leveraged further by T-cell checkpoint blockade, with immediate implications for clinical evaluation. Cancer Res; 76(13); 3929-41. ©2016 AACR.

Small Molecule Inhibition of MDM2–p53 Interaction Augments Radiation Response in Human Tumors

MDM2–p53 interaction and downstream signaling affect cellular response to DNA damage. AMG 232 is a potent small molecule inhibitor that blocks the interaction of MDM2 and p53. We examined the capacity of AMG 232 to augment radiation response across a spectrum of human tumor cell lines and xenografts. AMG 232 effectively inhibited proliferation and enhanced radiosensitivity via inhibition of damage repair signaling. Combined AMG 232 and radiation treatment resulted in the accumulation of γH2AX-related DNA damage and induction of senescence with promotion of apoptotic and/or autophagic cell death. Several molecules involved in senescence, autophagy, and apoptosis were specifically modulated following the combined AMG 232/radiation treatment, including FoxM1, ULK-1, DRAM, and BAX. In vivo xenograft studies confirmed more potent antitumor and antiangiogenesis efficacy with combined AMG 232/radiation treatment than treatment with drug or radiation alone. Taken together, these data identify the capacity of AMG 232 to augment radiation response across a variety of tumor types harboring functional p53. Mol Cancer Ther; 14(9); 1994–2003. ©2015 AACR.

Antitumor effects of MEHD7945A, a dual specific antibody against EGFR and HER3, in combination with radiation in lung and head and neck cancers

Human epidermal growth factor receptor family members (EGFR, HER2, HER3, and HER4) play important roles in tumorigenesis and response to cancer therapeutics. In this study, we evaluated the capacity of the dual-target antibody MEHD7945A that simultaneously targets EGFR and HER3 to modulate radiation response in lung and head and neck cancer models. Antitumor effects of MEHD7945A in combination with radiation were evaluated in cell culture and tumor xenograft models. Mechanisms that may contribute to increased radiation killing by MEHD7945A, including DNA damage and inhibition of EGFR–HER signaling pathways, were analyzed. Immunohistochemical analysis of tumor xenografts was conducted to evaluate the effect of MEHD7945A in combination with radiation on tumor growth and microenvironment. MEHD7945A inhibited basal and radiation-induced EGFR and HER3 activation resulting in the inhibition of tumor cell growth and enhanced radiosensitivity. MEHD7945A was more effective in augmenting radiation response than treatment with individual anti-EGFR or anti-HER3 antibodies. An increase in DNA double-strand breaks associated γ-H2AX was observed in cells receiving combined treatment with MEHD7945A and radiation. Immunohistochemical staining evaluation in human tumor xenografts showed that MEHD7945A combined with radiation significantly reduced the expression of markers of tumor proliferation and tumor vasculature. These findings reveal the capacity of MEHD7945A to augment radiation response in lung and head and neck cancers. The dual EGFR/HER3–targeting action of MEHD7945A merits further investigation and clinical trial evaluation as a radiation sensitizer in cancer therapy. Mol Cancer Ther; 14(9); 2049–59. ©2015 AACR.

Pan-HER Inhibitor Augments Radiation Response in Human Lung and Head and Neck Cancer Models

Purpose: Aberrant regulation of the EGF receptor family (EGFR, HER2, HER3, HER4) contributes to tumorigenesis and metastasis in epithelial cancers. Pan-HER represents a novel molecular targeted therapeutic composed of a mixture of six monoclonal antibodies against EGFR, HER2, and HER3. Experimental Design: In the current study, we examine the capacity of Pan-HER to augment radiation response across a series of human lung and head and neck cancers, including EGFR inhibitor–resistant cell lines and xenografts. Results: Pan-HER demonstrates superior antiproliferative and radiosensitizing impact when compared with cetuximab. The mechanisms underlying these effects appear to involve attenuation of DNA damage repair, enhancement of programmed cell death, cell-cycle redistribution, and induction of cellular senescence. Combined treatment of Pan-HER with single or fractionated radiation in human tumor xenografts reveals a potent antitumor and regrowth delay impact compared with Pan-HER or radiation treatment alone. Conclusions: These data highlight the capacity of Pan-HER to augment radiation response in lung and head and neck cancer models and support investigation of Pan-HER combined with radiation as a promising clinical therapeutic strategy. Clin Cancer Res; 22(3); 633–43. ©2015 AACR.

Tumor-Specific Inhibition of In Situ Vaccination by Distant Untreated Tumor Sites

Untreated tumor sites antagonize the systemic and local antitumor immune response to an in situ vaccination regimen. This effect is radiation sensitive and may be mediated by tumor-specific regulatory T cells harbored in the untreated tumor sites. In situ vaccination is an emerging cancer treatment strategy that uses local therapies to stimulate a systemic antitumor immune response. We previously reported an in situ vaccination effect when combining radiation (RT) with intratumor (IT) injection of tumor-specific immunocytokine (IC), a fusion of tumor-specific antibody and IL2 cytokine. In mice bearing two tumors, we initially hypothesized that delivering RT plus IT-IC to the “primary” tumor would induce a systemic antitumor response causing regression of the “secondary” tumor. To test this, mice bearing one or two syngeneic murine tumors of B78 melanoma and/or Panc02 pancreatic cancer were treated with combined external beam RT and IT-IC to the designated “primary” tumor only. Primary and secondary tumor response as well as animal survival were monitored. Immunohistochemistry and quantitative real-time PCR were used to quantify tumor infiltration with regulatory T cells (Treg). Transgenic “DEREG” mice or IgG2a anti–CTLA-4 were used to transiently deplete tumor Tregs. Contrary to our initial hypothesis, we observed that the presence of an untreated secondary tumor antagonized the therapeutic effect of RT + IT-IC delivered to the primary tumor. We observed reciprocal tumor specificity for this effect, which was circumvented if all tumors received RT or by transient depletion of Tregs. Primary tumor treatment with RT + IT-IC together with systemic administration of Treg-depleting anti–CTLA-4 resulted in a renewed in situ vaccination effect. Our findings show that untreated tumors can exert a tumor-specific, Treg-dependent, suppressive effect on the efficacy of in situ vaccination and demonstrate clinically viable approaches to overcome this effect. Untreated tumor sites antagonize the systemic and local antitumor immune response to an in situ vaccination regimen. This effect is radiation sensitive and may be mediated by tumor-specific regulatory T cells harbored in the untreated tumor sites. Cancer Immunol Res; 6(7); 825–34. ©2018 AACR.

Abstract 4495: Sym013, novel pan-HER monoclonal antibody mixture, augments radiation response in human lung and head and neck tumors

Sym013 represents a novel HER family targeting approach consisting of a mixture of six monoclonal antibodies against EGFR, HER2, and HER3. Sym013 effectively induces rapid, simultaneous down-regulation of all HER members. Compared with single receptor or dual receptor targeting of the HER family, Sym013 provides broader inhibition and greater down-regulation efficacy. In this current study, we examined the capacity of Sym013 in combination with ionizing radiation to augment tumor response in lung (NSCLC) and head and neck (HNSCC) cancer model systems. The anti-proliferative effects of Sym013 were confirmed showing 30-60% growth inhibition across a variety of NSCLC and HN 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4495. doi:10.1158/1538-7445.AM2014-4495

Abstract 2610: AMG 232, a small molecular inhibitor of MDM2 augments radiation response in human tumors harboring wild-type p53

Activation of p53 is an attractive therapeutic target in radiation oncology because of its tumor-suppressor activity. AMG 232 is an effective p53 activator via inhibition of p53 interaction with its primary negative regulator, MDM2. In the current study, we examine the capacity of AMG 232 to augment radiation response in a variety of human tumors derived from lung, breast, colorectal, melanoma and sarcoma. We first examined the anti-proliferative effect of AMG 232 and confirmed a dose-dependent growth inhibition following AMG 232 treatment across a panel of 7 cell lines harboring wild type p53. In addition, the lack of growth inhibition in a p53 null cell line H1299 confirmed the p53-dependent anti-proliferative effect of AMG 232. Using clonogenic survival analysis, we identified that treatment with AMG 232 significantly enhanced radiosensitivity in all cell lines tested. Immunoblot analysis revealed the capacity of AMG 232 to inhibit radiation-induced DNA damage repair via inhibition of ATM, DNAPK-DNA ligase IV and BRCA1-Rad51/52 pathways. Flow cytometric analysis also showed a significant accumulation of cell populations in G2/M phase and a strong increase in γH2AX expression at 48 hrs following 2 Gy of radiation and AMG 232 treatment. Consistently, AMG 232 was shown to activate 1433σ that blocked G2/M progression via inhibiting CDC2. Further imaging studies to detect senescence-associated β-galactosidase activity identified that combined treatment of AMG 232 and radiation significantly induced a senescence phenotype. These results suggest that AMG 232 augments radiation response via the induction of cell cycle arrest and/or senescence followed by cell death, likely reflecting inhibitory effects on DNA damage repair. In addition to increased apoptotic cell death following the combination treatment, we found that autophagy-related cell death may also be contributing to AMG 232-induced radiosensitivity. Finally, in a variety of human tumor xenograft models, we confirmed that the combination of AMG 232 and radiation resulted in a significant delay in tumor regrowth and superior anti-tumor efficacy than treatment with drug or radiation alone. Taken together, these data reveal the strong capacity of AMG 232 to augment radiation response across a variety of tumor types. These promising results provide a basis for the further investigation of MDM2 antagonists combined with radiation as a novel treatment approach in cancer therapy. Citation Format: Lauryn Werner, Shyhmin Huang, Eric A. Armstrong, Dave Francis, Tao Osgood, Jude Canon, Paul M. Harari. AMG 232, a small molecular inhibitor of MDM2 augments radiation response in human tumors harboring wild-type p53. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2610. doi:10.1158/1538-7445.AM2014-2610

Transcriptional-mediated effects of radiation on the expression of immune susceptibility markers in melanoma

BACKGROUND AND PURPOSE We recently reported a time-sensitive, cooperative, anti-tumor effect elicited by radiation (RT) and intra-tumoral-immunocytokine injection in vivo. We hypothesized that RT triggers transcriptional-mediated changes in tumor expression of immune susceptibility markers at delayed time points, which may explain these previously observed time-dependent effects. MATERIALS AND METHODS We examined the time course of changes in expression of immune susceptibility markers following in vitro or in vivo RT in B78 murine melanoma and A375 human melanoma using flow cytometry, immunoblotting, and qPCR. RESULTS Flow cytometry and immunoblot revealed time-dependent increases in expression of death receptors and T cell co-stimulatory/co-inhibitory ligands following RT in murine and human melanoma. Using high-throughput qPCR, we observed comparable time courses of RT-induced transcriptional upregulation for multiple immune susceptibility markers. We confirmed analogous changes in B78 tumors irradiated in vivo. We observed upregulated expression of DNA damage response markers days prior to changes in immune markers, whereas phosphorylation of the STAT1 transcription factor occurred concurrently with changes following RT. CONCLUSION This study highlights time-dependent, transcription-mediated changes in tumor immune susceptibility marker expression following RT. These findings may help in the design of strategies to optimize sequencing of RT and immunotherapy in translational and clinical studies.

Radiosensitization of adenoid cystic carcinoma with MDM2 inhibition

Purpose: Adenoid cystic carcinoma (ACC) is a rare cancer arising from the major or minor salivary gland tissues of the head and neck. There are currently no approved systemic agents or known radiosensitizers for ACC. Unlike the more common head and neck squamous cell carcinomas that frequently harbor TP53 mutations, ACCs contain TP53 mutations at a rate of <5%, rendering them an attractive target for MDM2 inhibition. Experimental Design: We report the successful establishment and detailed characterization of a TP53-WT ACC patient-derived xenograft (PDX), which retained the histologic features of the original patient tumor. We evaluated this model for response to the MDM2 inhibitor AMG 232 as monotherapy and in combination with radiotherapy. Results: AMG 232 monotherapy induced modest tumor growth inhibition, and radiation monotherapy induced a transient tumor growth delay in a dose-dependent fashion. Strikingly, combination treatment of AMG 232 with radiotherapy (including low-dose radiotherapy of 2 Gy/fraction) induced dramatic tumor response and high local tumor control rates 3 months following treatment. Posttreatment analysis revealed that although both AMG 232 and radiotherapy alone induced TP53 tumor-suppressive activities, combination therapy amplified this response with potent induction of apoptosis after combination treatment. Conclusions: These data identify that MDM2 inhibition can provide potent radiosensitization in TP53-WT ACC. In light of the absence of effective systemic agents for ACC, the powerful response profile observed here suggests that clinical trial evaluation of this drug/radiotherapy combination may be warranted to improve local control in this challenging malignancy. Clin Cancer Res; 23(20); 6044–53. ©2017 AACR.

Patient-Derived Adenoid Cystic Carcinoma Xenografts to Examine Personalized Radiation Therapy.

Purpose/Objective(s): Clinical radiotherapy has made significant advances since its inception, growing into a tertiary specialty with significant contributions to curative and palliative treatments of cancer and health care costs. A major limitation to its appropriate application, however, has been the lack of measurable biological indicators, or biomarkers that can reliably identify patients with cancers that are more or less likely to respond to these treatments. Materials/Methods: We conducted large-scale profiling of cellular survival after exposure to radiation in a diverse collection of 534 genetically annotated human tumor cell lines. Using data derived from a single, validated experimental platform we studied the genetic determinants of survival after radiation in 534 human cancer cell lines across 26 cancer types. We correlated radiation sensitivity and genomic parameters using the information-based similarity index, which is sensitive to non-linear relationships and offers better resolution at the high end of the matching range. Results: We showed that individual SCNA, gene mutations, and the basal expression of individual genes and gene sets correlate with radiation survival. By studying a large number of cancer types, we found that genetic correlates in any single cancer type can be found in other cancer types as well (e.g., Nrf2 activation in non-small cell lung cancer and hepatobiliary cancer and AR expression in prostate and breast adenocarcinomas). This supports the view that although diverse, cancer genomes reflect combinations of a limited number of functionally relevant events that can confer therapeutic resistance across cancer types. Conclusion: We identified several new genetic determinants of response to DNA damage that can have predictive capacity by identifying the likelihood of response to therapy and, consequently, prognosis. The potential for stratification of patients from heterogeneous populations to genetically similar subgroups can help guide the transition of radiotherapy from a generic population-based approach to one that is more personalized. Author Disclosure: B. Yard: None. D. Adams: None. P. Tamayo: None. P. Hammerman: None. M. Abazeed: None.