Restoration Of Tumor Suppressor Function Research Articles

Abstract 640: In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations

Abstract p53 tumor suppressor mutations result in loss of function of p53 in approximately 50% of all human cancers.TP53 missense, nonsense mutations and p53 deletion are present in about 50%, 12% and 5% of all p53 driven cancers, respectively. To date there is no effective treatment for rescuing p53 function in cancers using a pan-p53 approach. The use of ADGN-531 is a new potent strategy combining mRNA with a tumor selective peptide nanocarrier to rescue of p53 tumor suppressor function. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides in the form of nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring various p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and NSCLC NCI-H358 (p53 null/KRASG12C) mouse xenografts at a dose of 0.25-1.0 mg/kg weekly. ADGN-531/sotorasib synergy was evaluated in vitro on NCI-H358 (p53 null/KRASG12C) and Mia-PACA (p53R48W/KRASG12C) cells and in vivo on NCI-H358 mouse xenograft. ADGN-531 markedly inhibits the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations or p53-deletion, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition in SaOs2, SW403 and H358 xenografts independent of KRAS status. We demonstrated a strong synergy in a NSCLC H358 xenograft (tumor regression 80%) when restoring P53 function with ADGN-531 (0.5mg/kg, weekly) along with inhibition of KRASG12C mutant using sotorasib (10mg/kg, daily). ADGN-531 potentiated the effect of sotorasib on sotorasib resistant cells harboring high level of KRASG12C in permanent active state. ADGN-531 treatments are well tolerated, no sign of clinical toxicity was detected after single or repeated administrations. ADGN-531 is effective in rescuing p53 function both in vitro and in vivo across a wide range of p53 alterations. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy in P53 altered tumors independent of other driver mutations e.g., KRAS, or with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Veronique Josserand, Melanie Guidetti, Neil Desai. In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 640.

Abstract 550: p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors

Abstract The p53 tumor suppressor mutations resulting in loss of function control tumor proliferation in 50% of human cancers. TP53 missense and nonsense mutations are present in about 50% and 7-12% of the p53 driven cancer, respectively. To date there is no effective treatment for rescuing p53 function in cancers harboring p53 nonsense mutations. We have developed a new potent strategy combining mRNA with a tumor selective nanocarrier, to rescue of p53 tumor suppressor functions as potential therapeutic approach in cancers. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides that form stable neutral nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring different types of p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (weekly, 0.5 & 1.0mg/kg) was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and lung H358 (p53 null/KRASG12C) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment was evaluated on PARPi resistant SUM-149PT and OVCAR-8 and PARPi sensitive MDA-MB436 cells. ADGN-531/AMG-510 synergy was evaluated on H358 and M-PACA cells and H358 mouse xenograft. ADGN-531 markedly delay the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. ADGN-531 mediated p53 function rescue is directly correlated to the type of p53 mutation in the cancer cells. ADGN-531 induces 80% cell proliferation inhibition in p53 null/nonsense cells and 20 to 50% in p53 missense cells. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition. ADGN-531 (1.0mg/kg) induces 40-60% tumor regression SaOs2, SW403 and H358 xenografted mouse model. We demonstrated a synergistic combination between ADGN-531/AMG-510 a KRASG12C inhibitor in human lung carcinoma. ADGN-531 (0.5mg/kg, weekly)/AMG-510 (10mg/kg, daily) induces about 80% tumor regression, higher than with either single agents. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to Veliparib. ADGN-531 treatments are well tolerated, no sign of clinical toxicity or inflammatory response was detected after single or repeated administrations. ADGN-531 is effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy or in combination together with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Khadidja Boularache, Melanie Guidetti, Veronique Josserand, Neil Desai. p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 550.

62 (PB052) - p53 mRNA rescue of tumor suppressor function prevents tumor growth and restores PARPi sensitivity in p53-deficient cancers in vitro and in vivo

Background: Loss of function in tumor suppressor genes is commonly associated with the onset/progression of cancer and the development of therapeutic resistance. The p53 tumor suppressor mutations resulting in loss of function are common across variety of tumors and control tumor cell proliferation in more than 50% of cancers. P53 null mutations are present in about 10% of p53 driven cancers. However, to date there is no effective treatment for rescuing p53 function in cancers. We have developed a new potent strategy combing p53 mRNA with a tumor selective nanocarrier, to rescue p53 tumor suppressor function as potential therapeutic approach in cancers. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-531 nanoparticles containing full length p53-mRNA were evaluated on 20 different cancer cell lines (osteosarcoma, pancreas, colorectal, ovarian, lung, breast and prostate cancers) harboring different types of p53 mutations (null, deletion, nonsense, missense). Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry, Elisa and Tunel assay. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (0.5 mg/kg) was evaluated on colorectal SW403 (p53 deleted) and osteosarcoma SaOs2 (p53 null) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment in vitro was evaluated on PARPI resistant SUM-149PT and OVCAR-8 cells and on PARPi sensitive MDA-MB436 cells. Results: ADGN-531 NPs markedly delay the growth of a large panel of cancer cells harboring p53-null or nonsense mutations, by inducing cell cycle arrest in G1 due to p21 upregulation and apoptosis following PUMA activation. We demonstrated that ADGN-531 mediated p53 function rescue is directly correlated to the type and level of p53 mutation in the cancer cells. ADGN-531 induces 60% to 80% cell proliferation inhibition in p53 null or nonsense mutated cells and 20 to 50% in p53 missense mutated cells. Intravenous-administration ADGN-531 containing wild type p53-mRNA (0.5 mg/kg) resulted in tumor growth inhibition of 90% and 70% in SaOs2 and SW403 xenografted mouse model, respectively. ADGN-531 NPs treatments are well tolerated, without inducing clinical toxicity or inflammatory response. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to the PARPi veliparib. Conclusions: ADGN-531 nanoparticles are effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by the ADGN-531 NP targeted delivery strategy could be combined together with other therapies for potent combinatorial cancer treatment. No conflict of interest.

Abstract A144: Restoration of tumor suppressor function by mitochondrially targeting p14 peptide in tumor cells

Abstract Introduction Loss of CDKN2A (encoding p16INK4a and p14ARF) gene mainly by epigenetic alternations is associated with an increased susceptibility for development of malignant tumors. We previously reported the loss of the p14ARF function especially on the gefitinib-resistant lung adenocarcinomas (NSCLCs). In the present study, we identified the p14MIS (p14 minimal inhibitory sequence) peptide covering the functional core sequence of p14ARF, which is targeted to the mitochondria in the tumor cells and report its utility in growth suppression of biologically aggressive cancer cells of diverse origins. Experimental procedures Human tumor lines of various origins were treated with 10 μM of the p14MIS for 24 h or 48 h. The intracellular localization of p14MIS and growth suppression which is correlated to mitochondrial membrane potential by the p14MIS were examined by MTT assay and inverted fluorescence microscopy which is available to the peptide-treated living tumor cells. For in vivo study, the tumor xenograft mouse models were made by intraperitoneal injection of the human pancreatic adenocarcinoma cell, GFP-expressing BxPC3, and examined the antitumor effect of the peptide. The mice were daily treated with the 300ug/mouse of the p14MIS or with the polyarginine peptide (R9) as a control for a week, respectively. After the sequential treatment of the peptide, the mice of each group were autopsied and evaluated intraperitoneal dissemination (metastasis) of the GFP-expressing tumors. Results The p14MIS successfully targeted to the mitochondria and triggered an apoptosis to the various lineage of tumor cells by reduction of their mitochondrial membrane potential. The sensitivity and efficacy of the p14MIS was dependent on the expression level of endogenous mitochondrial ATPAF1 (F1-ATPase assembly protein) and also the magnitude of downregulation of the mitochondrial membrane potential in each tumor line. Moreover, in vivo delivery of the p14MIS to the pancreatic tumor model mice resulted in a 75% reduction in tumor volume compared with those in the control mice. Conclusions These results support the biological significance of the tumor suppressor molecule, p14ARF, especially localized at mitochondrias on tumor cells, and the utility of p14MIS peptide as an antitumor biotool against highly aggressive cancers. Although molecular details in tumor cells still remain to be clarified with respect to mitochondrial action of the p14MIS, it a potential of the peptide-based molecular therapy in intractable human malignancies of diverse origins. Citation Format: Ken Saito, Eisaku Kondo. Restoration of tumor suppressor function by mitochondrially targeting p14 peptide in tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A144.

A new molecular targeted therapeutic approach for renal cell carcinoma with a p16 functional peptide using a novel transporter system

Molecular targeting agents have become formidable anticancer weapons showing much promise against refractory tumors and functional peptides and are among the more desirable of these nanobio-tools. Intracellular delivery of multiple functional peptides forms the basis for a potent, non-invasive mode of delivery, providing distinctive therapeutic advantages. We examine the growth suppression efficiency of human renal cell carcinoma (RCC) by single-peptide targeting. We simultaneously introduced p16INK4a tumor suppressor peptides by Wr-T-mediated peptide delivery. Wr-T-mediated transport of p16INK4a functional peptide into 10 RCC lines, lacking expression of the p16INK4a molecule, reversed the specific loss of p16 function, thereby drastically inhibiting tumor growth in all but 3 lines by >95% within the first 96h. Invivo analysis using SK-RC-7 RCC xenografts in nude mice demonstrated tumor growth inhibition by the p16INK4a peptide alone, however, inoculation of Wr-T and the p16INK4a functional peptide mixture, via the heart resulted in complete tumor regression. Thus, restoration of tumor suppressor function with Wr-T peptide delivery represents a powerful approach, with mechanistic implications for the development of efficacious molecular targeting therapeutics against intractable RCC.

Abstract 610: Regression of spontaneous non-small cell lung cancers in P53 mutant transgenic mice following exposure to PRIMA-1

Abstract P53 mutations are common in lung cancer, thus restoration of p53 function in tumor cells have the potential to be an important approach for lung cancer therapy. The small molecule PRIMA-1 (p53-dependent reactivation and induction of massive apoptosis) has been shown to induce apoptosis in human tumor cells containing mutant p53 by restoration of tumor suppressor function of p53. This is believed to be mediated by a change in the conformation of mutated p53 protein, restoring DNA binding and activation of p53 target genes. We have developed a lung tumor model in transgenic mice in which the human mutant p53(273H) is expressed in a lung specific manner, under the control of the surfactant protein C promoter. These mice developed lung adenocarcinomas at an age range of 12-15 months. To evaluate feasibility and preliminary activity of PRIMA-1 on the murine spontaneous lung tumor, we treated SPC-p53-273H lung tumor bearing mice via intraperitoneal injection (i.p) with PRIMA-1 at a dose of 100 mg/kg in 0.2 ml PBS every other day for two weeks. Lung tumor bearing mice were identified with a micro-computed tomography (micro-CT) system and pre- and post-treatment tumor volumes were estimated based on the micro-CT image. Five mice received the PRIMA-1, and a control group of 4 lung tumor mice received PBS every other day for two weeks. A repeat CT scan was obtained the week after the last treatment. Results of post-treatment with pre-treatment CT volume comparisons are detailed below: These preliminary results indicate activity of PRIMA-1 in lung tumor bearing P53 mutant transgenics, and suggest the potential feasibility of using p53 restoring activity small molecules to treat lung cancer. Expansion of the sample size, as well as mechanistic and dosing studies is ongoing in order to strengthen the rationale to eventually pursue this strategy in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 610. doi:10.1158/1538-7445.AM2011-610

Molecular Approaches to Sarcoma Therapy

Soft tissue sarcomas comprise a heterogeneous group of aggressive tumors that have a relatively poor prognosis. Although conventional therapeutic regimens can effectively cytoreduce the overall tumor mass, they fail to consistently achieve a curative outcome. Alternative gene-based approaches that counteract the underlying neoplastic process by eliminating the clonal aberrations that potentiate malignant behavior have been proposed. As compared to the accumulation of gene alterations associated with epithelial carcinomas, sarcomas are frequently characterized by the unique presence of a single chromosomal translocation in each histological subtype. Similar to the Philadelphia chromosome associated with CML, these clonal abnormalities result in the fusion of two independent unrelated genes to generate a unique chimeric protein that displays aberrant activity believed to initiate cellular transformation. Secondary gene mutations may provide an additional growth advantage that further contributes to malignant progression. The recent clinical success of the tyrosine kinase inhibitor, STI571, suggests that therapeutic approaches specifically directed against essential survival factors in sarcoma cells may be effective. This review summarizes published approaches targeting a specific molecular mechanism associated with sarcomagenesis. The strategy and significance of published translational studies in six distinct areas are presented. These include: (1) the disruption of chimeric transcription factor activity; (2) inhibition of growth stimulatory post-translational modifications; (3) restoration of tumor suppressor function; (4) interference with angiogenesis; (5) induction of apoptotic pathways; and (6) introduction of toxic gene products. The potential for improving outcomes in sarcoma patients and the conceptual obstacles to be overcome are discussed.

Gene transfer of wild-type p53 results in restoration of tumor-suppressor function in a medulloblastoma cell line

The replacement of functional genes into cells that lack genes or have mutant genes is the basis of gene therapy. In cancer, where cells often have multiple genetic defects, the replacement of critical genes may suffice to suppress cell growth or induce cell death. The high frequency of mutations of the p53 tumor-suppressor gene in human cancers, including primary brain tumors, suggests that p53 plays a critical role in carcinogenesis and tumor progression. We report the successful transfer of the wild-type p53 gene using a defective herpes simplex viral vector into a human medulloblastoma cell line containing a mutant copy of p53. Upon gene transfer, we detected novel expression of wild-type p53 protein in the cells. In addition, the p53 protein was functionally active, since gene transfer resulted in increased levels of mdm2 proteins and induced cell cycle arrest of the majority of transduced cells. To our knowledge, this is the first report of the use of this vector system to carry wild-type p53. We conclude that defective herpes simplex viral vectors can transfer and express p53 in human primary brain tumor cells in vitro, restoring wild-type p53 tumor-suppressor functions.