Evaluation of tissue- and plasma-derived tumor mutational burden (TMB) and genomic alterations of interest in CheckMate 848, a study of nivolumab combined with ipilimumab and nivolumab alone in patients with advanced or metastatic solid tumors with high TMB.

BACKGROUND: An accumulation of somatic mutations in tumors leads to increased neoantigen levels and antitumor immune response. Tumor mutational burden (TMB) reflects the rate of somatic mutations in the tumor genome, as determined from tumor tissue (tTMB) or blood (bTMB). While high tTMB is a biomarker of immune checkpoint inhibitor (ICI) treatment efficacy, few studies have explored the clinical utility of bTMB, a less invasive alternative for TMB assessment. Establishing the correlation between tTMB and bTMB would provide insight into whether bTMB is a potential substitute for tTMB. We explored the tumor genomes of patients enrolled in CheckMate 848 with measurable TMB. The correlation between tTMB and bTMB, and the factors affecting it, were evaluated. METHODS: In the phase 2 CheckMate 848 (NCT03668119) study, immuno-oncology-naïve patients with advanced, metastatic, or unresectable solid tumors and tTMB-high or bTMB-high (≥10 mut/Mb) were prospectively randomized 2:1 to receive nivolumab plus ipilimumab or nivolumab monotherapy. Tissue and plasma DNA sequencing was performed using the Foundation Medicine FoundationOne CDx and bTMB Clinical Trial Assays, respectively. tTMB was quantified from coding variants, insertions, and deletions, and bTMB from somatic base substitutions. Correlations between tTMB and bTMB were determined across samples and with respect to maximum somatic allele frequency (MSAF). Assay agreement and variant composition were also evaluated. RESULTS: A total of 1,438 and 1,720 unique tissue and blood samples, respectively, were obtained from 1,954 patients and included >100 screened disease ontologies, with 1,017 unique pairs of tTMB and bTMB measurements available for assessment. Median tTMB and bTMB were 3.8 and 3.5 mut/Mb, respectively. A significant correlation between tTMB and bTMB (r=0.48, p<0.0001) was observed across all sample pairs, which increased to r=0.54 (p<0.0001) for samples with MSAF≥1%. Assay concordance was highest for samples with MSAF≥10% across multiple disease ontologies and observed for both responders and non-responders to ICI therapy. The variants contributing to tTMB and bTMB were similar. CONCLUSIONS: We observed that tTMB and bTMB had a statistically significant correlation, particularly for samples with high MSAF, and that this correlation applied across disease ontologies. Further investigation into the clinical utility of bTMB is warranted.

Addressing resistance to PD-1/PD-(L)1 pathway inhibition: considerations for combinatorial clinical trial designs.

With multiple PD-(L)1 inhibitors approved across dozens of indications by the US Food and Drug Administration, the number of patients exposed to these agents in adjuvant, first-line metastatic, second-line metastatic, and refractory treatment settings is increasing rapidly. Although some patients will experience durable benefit, many have either no clinical response or see their disease progress following an initial response to therapy. There is a significant need to identify therapeutic approaches to overcome resistance and confer clinical benefits for these patients. PD-1 pathway blockade has the longest history of use in melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC). Therefore, these settings also have the most extensive clinical experience with resistance. In 2021, six non-profit organizations representing patients with these diseases undertook a year-long effort, culminating in a 2-day workshop (including academic, industry, and regulatory participants) to understand the challenges associated with developing effective therapies for patients previously exposed to anti-PD-(L)1 agents and outline recommendations for designing clinical trials in this setting. This manuscript presents key discussion themes and positions reached through this effort, with a specific focus on the topics of eligibility criteria, comparators, and endpoints, as well as tumor-specific trial design options for combination therapies designed to treat patients with melanoma, NSCLC, or RCC after prior PD-(L)1 pathway blockade.

Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism.

Prohibitin is a growth-suppressive protein that has multiple functions in the nucleus and the mitochondria. Our earlier studies had shown that prohibitin represses the activity of E2F transcription factors while enhancing p53-mediated transcription. At the same time, prohibitin has been implicated in mediating the proper folding of mitochondrial proteins. We had found that treatment of cells with camptothecin, a topoisomerase 1 inhibitor, led to the export of prohibitin and p53 from the nucleus to the mitochondria. Here we show that the camptothecin-induced export of prohibitin occurs preferentially in transformed cell lines, but not in untransformed or primary cells. Cells that did not display the translocation of prohibitin were refractive to the apoptotic effects of camptothecin. The translocation was mediated by a putative nuclear export signal at the C-terminal region of prohibitin; fusion of the nuclear export signal (NES) of prohibitin to green fluorescence protein led to its export from the nucleus. Leptomycin B could inhibit the nuclear export of prohibitin showing that it was a CRM-1-dependent event driven by Ran GTPase. Confirming this, prohibitin was found to physically interact with CRM-1, and this interaction was significantly higher in transformed cells. Delivery of a peptide corresponding to the NES of prohibitin prevented the export of prohibitin to cytoplasm and protected cells from apoptosis. These results suggest that the regulated translocation of prohibitin from the nucleus to the mitochondria facilitates its pleiotropic functions and might contribute to its anti-proliferative and tumor suppressive properties.

Disruption of the Rb--Raf-1 interaction inhibits tumor growth and angiogenesis.

The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G(1) phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.

Prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling.

Prohibitin, a potential tumor suppressor protein, has been shown to inhibit cell proliferation and repress E2F transcriptional activity. Though prohibitin has potent transcriptional functions in the nucleus, a mitochondrial role for prohibitin has also been proposed. Here we show that prohibitin is predominantly nuclear in two breast cancer cell lines where it co-localizes with E2F1 and p53. Upon apoptotic stimulation by camptothecin, prohibitin is exported to perinuclear regions where it localizes to mitochondria. The data presented here also show that prohibitin is capable of physically interacting with p53 in vivo and in vitro. Prohibitin was found to enhance p53-mediated transcriptional activity and cotransfection of an antisense prohibitin construct reduces p53-mediated transcriptional activation. Prohibitin appears to induce p53-mediated transcription by enhancing its recruitment to promoters, as detected by chromatin immunoprecipitation assays. These results suggest that prohibitin is capable of modulating Rb/E2F as well as p53 regulatory pathways.

Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression.

The potential tumor suppressor protein prohibitin can prevent cell proliferation and this required its binding to the Rb protein. Prohibitin could repress the transcriptional activity of E2F family members and this required a part of the marked box region of E2F. The sub-cellular localization of prohibitin has been variously attributed to the mitochondria as well as the inner cell membrane. Here we show that a subset of prohibitin molecules are present in the nucleus where it co-localizes with the Rb protein. Deletion of a putative amino-terminal membrane-docking domain of prohibitin had no effect on its ability to suppress cell proliferation or inhibit E2F activity. Our experiments show that a 53 amino-acid stretch of E2F1 is sufficient for being targeted by prohibitin; fusion of this region to GAL4-VP16 construct could make it susceptible to prohibitin-mediated, but not Rb-mediated repression. Prohibitin, like Rb, could repress transcription from SV40 and major late promoters when recruited directly to DNA. Prohibitin mediated transcriptional repression required histone-deacetylase activity, but unlike Rb, additional co-repressors like N-CoR are also involved. Repression by prohibitin correlates with histone deacetylation on promoters and this was reversed by IgM stimulation of cells; IgM did not affect Rb-mediated repression or deacetylation of the promoters. Prohibitin thus appears to repress E2F-mediated transcription utilizing different molecular mediators and facilitate channeling of specific signaling pathways to the cell cycle machinery.

Differential regulation of Rb family proteins and prohibitin during camptothecin-induced apoptosis.

Prohibitin, a potential tumor suppressor, is known to induce growth suppression and repress E2F-mediated transcription. These growth regulatory functions of prohibitin require a physical interaction with the Rb protein. We now find that prohibitin protects cells from apoptosis mediated by camptothecin, a topoisomerase I inhibitor. Camptothecin treatment of Ramos B cells leads to the degradation of Rb protein and phosphorylation of its family members, p107 and p130. This correlates with an increase in the levels of cyclin E as well as the kinase activity associated with it. Inactivation of Rb leads to the dissociation and release of free E2F. We find also that E2F activity is induced upon camptothecin treatment, but this increase is absent in prohibitin overexpressing cells. It thus appears that prohibitin may be inhibiting apoptosis by downregulating E2F activity when Rb family members are inactive.