The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1(ΔG)) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1(ΔG/ΔG) mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1(ΔG/ΔG) MEFs display a magnitude of E2F target gene derepression similar to that of Rb1(-/-) cells, even though Rb1(ΔG/ΔG) cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1(ΔG/ΔG) MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1(ΔG/ΔG) mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.
Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes.
The mammalian G1-S phase transition is controlled by the opposing forces of cyclin-dependent kinases (CDK) and the retinoblastoma protein (pRB). Here, we present evidence for systems-level control of cell cycle arrest by pRB-E2F and p27-CDK regulation. By introducing a point mutant allele of pRB that is defective for E2F repression (Rb1G) into a p27KIP1 null background (Cdkn1b-/-), both E2F transcriptional repression and CDK regulation are compromised. These double-mutant Rb1G/G; Cdkn1b-/- mice are viable and phenocopy Rb1+/- mice in developing pituitary adenocarcinomas, even though neither single mutant strain is cancer prone. Combined loss of pRB-E2F transcriptional regulation and p27KIP1 leads to defective proliferative control in response to various types of DNA damage. In addition, Rb1G/G; Cdkn1b-/- fibroblasts immortalize faster in culture and more frequently than either single mutant genotype. Importantly, the synthetic DNA damage arrest defect caused by Rb1G/G; Cdkn1b-/- mutations is evident in the developing intermediate pituitary lobe where tumors ultimately arise. Our work identifies a unique relationship between pRB-E2F and p27-CDK control and offers in vivo evidence that pRB is capable of cell cycle control through E2F-independent effects.
RB-E2F transcriptional control plays a key role in regulating the timing of cell cycle progression from G1 to S-phase in response to growth factor stimulation. Despite this role, it is genetically dispensable for cell cycle exit in primary fibroblasts in response to growth arrest signals. Mice engineered to be defective for RB-E2F transcriptional control at cell cycle genes were also found to live a full lifespan with no susceptibility to cancer. Based on this background we sought to probe the vulnerabilities of RB-E2F transcriptional control defects found in Rb1R461E,K542E mutant mice (Rb1G) through genetic crosses with other mouse strains. We generated Rb1G/G mice in combination with Trp53 and Cdkn1a deficiencies, as well as in combination with KrasG12D. The Rb1G mutation enhanced Trp53 cancer susceptibility, but had no effect in combination with Cdkn1a deficiency or KrasG12D. Collectively, this study indicates that compromised RB-E2F transcriptional control is not uniformly cancer enabling, but rather has potent oncogenic effects when combined with specific vulnerabilities.
Abstract AVID100 is a novel rationally designed antibody drug conjugate (ADC) that specifically targets the epidermal growth factor receptor (EGFR). EGFR is highly expressed on a variety of cancers making it a promising target for ADCs. However, due to the presence of EGFR on normal skin cells, on-target off-tumor toxicity is a concern. AVID100 is an anti-EGFR-DM1 conjugate exhibiting high potency against cancer cells relative to unconjugated antibodies, while not exhibiting increased toxicity on normal cells. In a series of in vitro and in vivo experiments, we provide mechanistic evidence rationalizing why AVID100 shows higher cytotoxicity on tumor cells compared to normal cells. The antibody moiety of AVID100, denoted MAB100, was shown to have a high affinity for EGFR (approximately 2nM) and to compete with EGF for binding to the EGFR. Also, MAB100 prevented downstream signalling from EGFR. In addition to exhibiting full antagonist activity, MAB100 effectively delivered the microtubule inhibitor DM1 to tumor cells, as demonstrated by an increase in apoptosis in AVID100-treated tumor cells relative to MAB100-treated tumor cells. AVID100 was very effective on tumor cell lines derived from breast, head and neck, and lung cancers with the cytotoxic IC50 values generally correlating with the number of EGFR molecules on the cell surface. Importantly, on keratinocytes, AVID100 did not exhibit increased cytotoxicity relative to MAB100. We hypothesized that this lack of increase in cytotoxicity on keratinocytes is due to the antagonistic effect of MAB100 on EGFR signalling. Blocking the EGFR pathway in keratinocytes strongly inhibits their proliferation, which should protect them against the cytotoxic action of DM1. To address this hypothesis, we compared the effect of AVID100 to that of a non-antagonistic anti-EGFR ADC, huML66-DM1. As expected, both ADCs were highly effective at killing MDA-MB-468 cancer cells, confirming their ability to deliver DM1. In contrast, keratinocytes exhibited a significantly higher level of apoptosis in response to huML66-DM1 as compared to AVID100. These results confirm that the antagonistic nature of the antibody moiety of AVID100 plays a critical role in protecting keratinocytes from the cytotoxic effect of DM1. Finally, the activity of AVID100 was investigated in multiple mouse xenograft studies, including in MDA-MB-468 human breast adenocarcinoma, H292 NSCLC, and FaDu SSCHN models. AVID100 treatment significantly reduced tumor growth and caused tumor regressions in some of the mice, even when administered as a single dose. Toxicology studies in cynomolgus monkeys demonstrated that AVID100 was well tolerated at up to 4 weekly doses of 10mg/kg. AVID100 has completed a Phase 1 clinical trial with the recommended Phase 2 dose having been determined. The Phase 2 trial of AVID100 is ongoing. Citation Format: Michael J. Thwaites, Rene Figueredo, Gilles Tremblay, James Koropatnick, Victor Goldmacher, Maureen O’Connor-McCourt. AVID100 is an anti-EGFR ADC that promotes DM1-meditated cytotoxicity on cancer cells but not on normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 218.
