MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Evidence that the human cell cycle is a series of uncoupled, memoryless phases.

The cell cycle is canonically described as a series of four consecutive phases: G1, S, G2, and M. In single cells, the duration of each phase varies, but the quantitative laws that govern phase durations are not well understood. Using time-lapse microscopy, we found that each phase duration follows an Erlang distribution and is statistically independent from other phases. We challenged this observation by perturbing phase durations through oncogene activation, inhibition of DNA synthesis, reduced temperature, and DNA damage. Despite large changes in durations in cell populations, phase durations remained uncoupled in individual cells. These results suggested that the independence of phase durations may arise from a large number of molecular factors that each exerts a minor influence on the rate of cell cycle progression. We tested this model by experimentally forcing phase coupling through inhibition of cyclin-dependent kinase 2 (CDK2) or overexpression of cyclin D. Our work provides an explanation for the historical observation that phase durations are both inherited and independent and suggests how cell cycle progression may be altered in disease states.

Next-generation Tumor-homing Induced Neural Stem Cells as an Adjuvant to Radiation for the Treatment of Metastatic Lung Cancer.

The spread of non-small cell lung cancer (NSCLC) to the leptomeninges is devastating with a median survival of only a few months. Radiation offers symptomatic relief, but new adjuvant therapies are desperately needed. Spheroidal, human induced neural stem cells (hiNeuroS) secreting the cytotoxic protein, TRAIL, have innate tumoritropic properties. Herein, we provide evidence that hiNeuroS-TRAIL cells can migrate to and suppress growth of NSCLC metastases in combination with radiation. In vitro cell tracking and post-mortem tissue analysis showed that hiNeuroS-TRAIL cells migrate to NSCLC tumors. Importantly, isobolographic analysis suggests that TRAIL with radiation has a synergistic cytotoxic effect on NSCLC tumors. In vivo, mice treated with radiation and hiNeuroS-TRAIL showed significant (36.6%) improvements in median survival compared to controls. Finally, bulk mRNA sequencing analysis showed both NSCLC and hiNeuroS-TRAIL cells showed changes in genes involved in migration following radiation. Overall, hiNeuroS-TRAIL cells +/- radiation have the capacity to treat NSCLC metastases.

Immune-Mediated Effects of Microplanar Radiotherapy with a Small Animal Irradiator.

Spatially fractionated radiotherapy has been shown to have effects on the immune system that differ from conventional radiotherapy (CRT). We compared several aspects of the immune response to CRT relative to a model of spatially fractionated radiotherapy (RT), termed microplanar radiotherapy (MRT). MRT delivers hundreds of grays of radiation in submillimeter beams (peak), separated by non-radiated volumes (valley). We have developed a preclinical method to apply MRT by a commercial small animal irradiator. Using a B16-F10 murine melanoma model, we first evaluated the in vitro and in vivo effect of MRT, which demonstrated significant treatment superiority relative to CRT. Interestingly, we observed insignificant treatment responses when MRT was applied to Rag-/- and CD8-depleted mice. An immuno-histological analysis showed that MRT recruited cytotoxic lymphocytes (CD8), while suppressing the number of regulatory T cells (Tregs). Using RT-qPCR, we observed that, compared to CRT, MRT, up to the dose that we applied, significantly increased and did not saturate CXCL9 expression, a cytokine that plays a crucial role in the attraction of activated T cells. Finally, MRT combined with anti-CTLA-4 ablated the tumor in half of the cases, and induced prolonged systemic antitumor immunity.

Dual inhibition of DNA-PK and DNA polymerase theta overcomes radiation resistance induced by p53 deficiency.

TP53 deficiency in cancer is associated with poor patient outcomes and resistance to DNA damaging therapies. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Using live cell imaging of DNA double-strand breaks (DSBs) and cell cycle state transitions, we show that p53-deficient cells exhibit accelerated repair of radiomimetic-induced DSBs arising in S phase. Low-dose DNA-dependent protein kinase (DNA-PK) inhibition increases the S-phase DSB burden in p53-deficient cells, resulting in elevated rates of mitotic catastrophe. However, a subset of p53-deficient cells exhibits intrinsic resistance to radiomimetic-induced DSBs despite DNA-PK inhibition. We show that p53-deficient cells under DNA-PK inhibition utilize DNA polymerase theta (Pol θ)-mediated end joining repair to promote their viability in response to therapy-induced DSBs. Pol θ inhibition selectively increases S-phase DSB burden after radiomimetic therapy and promotes prolonged G2 arrest. Dual inhibition of DNA-PK and Pol θ restores radiation sensitivity in p53-deficient cells as well as in p53-mutant breast cancer cell lines. Thus, combination targeting of DNA-PK- and Pol θ-dependent end joining repair represents a promising strategy for overcoming resistance to DNA damaging therapies in p53-deficient cancers.

A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability.

The Mre11-Rad50-Nbs1 complex is a DNA double-strand break sensor that mediates a tumor-suppressive DNA damage response (DDR) in cells undergoing oncogenic stress, yet the mechanisms underlying this effect are poorly understood. Using a genetically inducible primary mammary epithelial cell model, we demonstrate that Mre11 suppresses proliferation and DNA damage induced by diverse oncogenic drivers through a p53-independent mechanism. Breast tumorigenesis models engineered to express a hypomorphic Mre11 allele exhibit increased levels of oncogene-induced DNA damage, R-loop accumulation, and chromosomal instability with a characteristic copy number loss phenotype. Mre11 complex dysfunction is identified in a subset of human triple-negative breast cancers and is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3 related (ATR) and poly (ADP-ribose) polymerase (PARP). Thus, deficiencies in the Mre11-dependent DDR drive proliferation and genome instability patterns in p53-deficient breast cancers and represent an opportunity for therapeutic exploitation.

Genetic determinants of cellular addiction to DNA polymerase theta.

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.