a b s t r a c tThe role of p53 as the ''guardian of the genome'' in differentiated somatic cells, triggering various biological processes, is well established. Recent studies in the stem cell field have highlighted a profound role of p53 in stem cell biology as well. These studies, combined with basic data obtained 20 years ago, provide insight into how p53 governs the quantity and quality of various stem cells, ensuring a sufficient repertoire of normal stem cells to enable proper development, tissue regeneration and a cancer free life. In this review we address the role of p53 in genomically stable embryonic stem cells, a unique predisposed cancer stem cell model and adult stem cells, its role in the generation of induced pluripotent stem cells, as well as its role as the barrier to cancer stem cell formation.

1,5 p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.

While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins that can be deconvoluted using common microscopy equipment. To address this limitation, we develop visual barcodes that discriminate the clonal identity of single cells by different fluorescent proteins that are targeted to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then use visual barcodes to generate ‘Signalome’ cell-lines by mixing 12 clones of different live reporters into a single population, allowing simultaneous monitoring of the activity in 12 branches of signaling, at clonal resolution, over time. Using the ‘Signalome’ we identify two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological systems.

While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins that can be deconvoluted using common microscopy equipment. To address this limitation we developed visual barcodes that discriminate the clonal identity of single cells by targeting different fluorescent proteins to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then used visual barcodes to generate ‘Signalome’ cell-lines by multiplexing live reporters to monitor the simultaneous activity in 12 branches of signaling, in live cells, at single cell resolution, over time. Using the ‘Signalome’ we identified two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological system

Background: Margetuximab (M) has demonstrated anti-tumor activity in patients with advanced HER2+ gastric cancer and PFS superiority to trastuzumab (T) in pre-treated metastatic HER2+ breast cancer patients (1). Similar to T, M inhibits HER2 signaling; additionally, M enhances engagement with the activating Fcγ receptor (FcR)IIIA, while diminishing interaction with the inhibitory FcRIIB. Given the role of FcRs in coupling innate and adaptive immune responses, clinical studies of M in combination with PD-1 or PD-1 plus LAG-3 blockade have been initiated (2,3). The combination of M with tebotelimab, an investigational bispecific DART® molecule blocking PD-1 and LAG-3, has demonstrated encouraging early evidence of anti-tumor activity in patients with advanced HER2+ malignancies, including those with PD-L1-negative tumors (3). Here we present data supporting the potential mechanisms underlying the anti-tumor activity elicited by M and tebotelimab combination therapy. Methods: PBMCs were challenged with HER2-expressing tumor cell lines in the presence of M or T. Immune modulation was assessed by analysis of immune cell phenotype, gene expression profiles, and cytokine secretome. Functional activity of immune cells following exposure to the combination of M with tebotelimab was evaluated. Results: M induced greater upregulation relative to T of both co-stimulatory (eg 4-1BB) and/or checkpoint molecules (eg PD-L1 and LAG-3) on NK cells and/or monocytes in PBMCs cocultured with HER2-expressing tumor cells. Compared with T, M also induced greater proinflammatory cytokine release, including IFNγ that in turn upregulates PD-L1 and MHC class I expression on co-cultured tumor cells. These effects were not observed with an Fc-null version of M or were inhibited by the addition of FcR-blocking Abs, demonstrating Fc:FcR dependency of M-induced immune activation. Interestingly, CD8 T cells, which lack FcR, also exhibited LAG-3 upregulation in the above cultures, suggesting the effects of M can also be mediated via a cascade mechanism. An IFNγ neutralizing mAb reduced tumor cell-associated PD-L1 and MHC class I upregulation, but not LAG-3 induction on T-cells. PBMCs, pre-treated with a combination of M and tebotelimab in the presence of HER2-expressing tumor cells, demonstrated enhanced cytolytic activity against both the canonical NK cell target, K562, or M-opsonized HER2-positive tumor cells. Conclusions: M, through an Fc-dependent mechanism, can upregulate checkpoint molecules on NK-cells, CD8 T cells and tumor cells, thereby sensitizing them to immune checkpoint blockade. In turn, PD-1 and LAG-3 blockade by tebotelimab enhances M-mediated NK cell cytolytic activity in vitro. References: 1. Rugo et al., 2019 ASCO oral presentation 2. Catenacci et al., 2020 Lancet Oncology 21(8):1066-76 3. Luke et al., 2020 ASCO oral presentation Citation Format: Xiaoyu Zhang, Haiquan Li, Yinhua Yang, Qihong Xu, Tom Kaufman, Doug Smith, Jeffrey Nordstrom, Ezio Bonvini, Paul Moore. Enhanced HER2-dependent immune activation by margetuximab, an investigational Fc-engineered anti-HER2 mAb, supports combination with checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1555.

p53 loss of heterozygosity (LOH) is a frequent event in tumors of somatic and Li-Fraumeni syndrome patients harboring p53 mutation. Here, we focused on resolving a possible crosstalk between the immune-system and p53 LOH. Previously, we reported that p53 heterozygous bone-marrow mesenchymal progenitor cells undergo p53 LOH in-vivo. Surprisingly, the loss of either the wild-type p53 allele or mutant p53 allele was detected with a three-to-one ratio in favor of losing the mutant allele. In this study, we examined whether the immune-system can affect the LOH directionality in bone marrow progenitors. We found that mesenchymal progenitor cells derived from immune-deficient mice exhibited the same preference of losing the mutant p53 allele as immune-competent matched cells, nevertheless, these animals showed a significantly shorter tumor-free survival, indicating the possible involvement of immune surveillance in this model. Surprisingly, spontaneous tumors of p53 heterozygous immune-deficient mice exhibited a significantly higher incidence of p53 LOH compared to that observed in tumors derived of p53 heterozygous immune-competent mice. These findings indicate that the immune-system may affect the p53 LOH prevalence in spontaneous tumors. Thus suggesting that the immune-system may recognize and clear cells that underwent p53 LOH, whereas in immune-compromised mice, those cells will form tumors with shorter latency. In individuals with a competent immune-system, p53 LOH independent pathways may induce malignant transformation which requires a longer tumor latency. Moreover, this data may imply that the current immunotherapy treatment aimed at abrogating the inhibition of cellular immune checkpoints may be beneficial for LFS patients.