Chromosomal Instability
The Maciejowski studies mechanisms underlying genomic instability and innate immunity at nuclear aberrations, such as micronuclei (MN) and chromosome bridges. This current focus is based on John's postdoctoral work in Titia de Lange’s lab, where he discovered that dicentric chromosomes formed by telomere fusion persist through mitosis to form chromosome bridges that are ultimately shattered leading to clustered mutation. The lab discovered that the ER-anchored nuclease TREX1 damages cytosol-exposed DNA at MN and chromosome bridges and limits activation of the pro-inflammatory DNA sensor cGAS at these nuclear aberrations. Surprisingly, our data also show that TREX1 depends on its association with the ER to catalyze DNA damage and inhibit cGAS activation at MN. My lab now seeks to 1. Determine the mechanisms of ER-dependent TREX1 recruitment to MN, 2. Dissect the role of TREX1-interaction partners and non-catalytic elements of TREX1 in cGAS-STING regulation 3. Pursue therapeutic opportunities arising from TREX1 MN attack. Our long-term goals are to identify strategies to improve immunotherapies and obtain fundamental insights into cancer genome evolution.
ER-directed genomic instability & immune control at sites of nuclear envelope rupture
We discovered that ER-directed resection of micronuclear DNA by TREX1 is a critical regulator of cytosolic DNA sensing in chromosomally unstable cells. Using a novel method for purifying MN, we found that TREX1 stably associates with ruptured MN where it damages cytosol-exposed micronuclear DNA and inhibits cGAS activation. Live-cell imaging and immunofluorescence-based assays confirm that TREX1 rapidly accumulates at MN and chromosome bridges after nuclear envelope rupture and damages micronuclear DNA. Surprisingly, TREX1 affinity for DNA is dispensable for its localization, arguing against a model in which cytosol-exposed chromatin directly attracts TREX1. Instead, the ER plays a critical role in directing TREX1 localization and activity at MN. Frameshift mutations in TREX1, previously associated with autoimmune diseases, truncates the C-terminal region (CTR) of the protein, deletes the predicted ER transmembrane domain, disrupts TREX1 association with the ER, compromises TREX1 activity in ruptured MN, and relieves cGAS inhibition. Analysis of a chimeric protein where the TREX1 CTR is replaced with the transmembrane domain of the core ER transporter protein Sec61 confirmed that ER-tethering is critical for TREX1 activity and cGAS inhibition at ruptured MN. Together, our results identify TREX1 as a critical regulator of immune sensing in chromosomally unstable cells, define new substrates of TREX1 relevant to disease, and establish ER-tethering as a critical director of TREX1 nucleolytic activity. These findings also provide a mechanistic basis for the importance of TREX1 ER-tethering in preventing autoimmunity. Read more here.
Intratumoral TREX1 induction promotes immune evasion
Chromosomal instability is a hallmark of human cancer that is associated with aggressive disease characteristics. Chromosome mis-segregations help fuel natural selection, but they risk provoking a cGAS-STING immune response through the accumulation of cytosolic DNA. The mechanisms of how tumors benefit from chromosomal instability while mitigating associated risks, such as enhanced immune surveillance, are poorly understood. We identified cGAS-STING-dependent upregulation of the nuclease TREX1 as an adaptive, negative feedback mechanism that promotes immune evasion through digestion of cytosolic DNA. TREX1 loss diminishes tumor growth, prolongs survival of host animals, increases tumor immune infiltration, and potentiates response to immune checkpoint blockade selectively in tumors capable of mounting a type I interferon response downstream of STING. Together, these data demonstrate that TREX1 induction shields chromosomally unstable tumors from immune surveillance by dampening type I interferon production and suggest that TREX1 inhibitors might be used to selectively target tumors that have retained the inherent ability to mount an interferon response downstream of STING. Read more here.
TREX1 promotes chromosome fragmentation during telomere crisis
Our previous work suggested that chromothripsis and kataegis may arise from chromosome bridges formed during telomere crisis, a period of genome instability that occurs during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes. We have now used our in vitro telomere crisis model to examine the mechanisms underlying chromothripsis and kataegis. We focused on TREX1 as our previous data showed that TREX1 resects chromosome bridge DNA to promote their resolution. To determine whether TREX1 contributes to chromothripsis after telomere crisis, TREX1-deficient cell lines generated by CRISPR-Cas9 editing (hereafter TREX1 KOs) were subjected to telomere crisis alongside TREX1-proficient counterparts and clonal post-crisis descendants were isolated for WGS. Comparison of low-coverage WGS data obtained from 417 TREX1 KO post-crisis clones with 117 TREX1-proficient cells showed that the frequency of complex events was lower in the TREX1 KO setting both with regard to clones containing complex events and the proportion of chromosomes showing complex events. Furthermore, the number of copy number changes associated with complex events was lower in the TREX1 KO setting. Selection of 14 wild-type postcrisis clones for high coverage WGS revealed that 12 exhibited rearrangement patterns consistent with chromothripsis. In contrast, chromothripsis-associated patterns were only detected in ~3% of the 14 TREX1 KO clones. These data indicate that TREX1 plays a critical role in the chromothripsis that results from DNA bridge resolution. Read more here.