Transient genomic instability drives tumorigenesis through accelerated clonal evolution.

Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.

Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin ß1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.

Chronic centrosome amplification without tumorigenesis.

Centrosomes are microtubule-organizing centers that facilitate bipolar mitotic spindle assembly and chromosome segregation. Recognizing that centrosome amplification is a common feature of aneuploid cancer cells, we tested whether supernumerary centrosomes are sufficient to drive tumor development. To do this, we constructed and analyzed mice in which centrosome amplification can be induced by a Cre-recombinase-mediated increase in expression of Polo-like kinase 4 (Plk4). Elevated Plk4 in mouse fibroblasts produced supernumerary centrosomes and enhanced the expected mitotic errors, but proliferation continued only after inactivation of the p53 tumor suppressor. Increasing Plk4 levels in mice with functional p53 produced centrosome amplification in liver and skin, but this did not promote spontaneous tumor development in these tissues or enhance the growth of chemically induced skin tumors. In the absence of p53, Plk4 overexpression generated widespread centrosome amplification, but did not drive additional tumors or affect development of the fatal thymic lymphomas that arise in animals lacking p53. We conclude that, independent of p53 status, supernumerary centrosomes are not sufficient to drive tumor formation.

Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.

Macrophage migration inhibitory factor as a chaperone inhibiting accumulation of misfolded SOD1.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons and accompanied by accumulation of misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and the endoplasmic reticulum (ER). Using inhibition of misfolded SOD1 deposition onto mitochondria as an assay, a chaperone activity abundant in nonneuronal tissues is now purified and identified to be the multifunctional macrophage migration inhibitory factor (MIF), whose activities include an ATP-independent protein folding chaperone. Purified MIF is shown to directly inhibit mutant SOD1 misfolding. Elevating MIF in neuronal cells suppresses accumulation of misfolded SOD1 and its association with mitochondria and the ER and extends survival of mutant SOD1-expressing motor neurons. Accumulated MIF protein is identified to be low in motor neurons, implicating correspondingly low chaperone activity as a component of vulnerability to mutant SOD1 misfolding and supporting therapies to enhance intracellular MIF chaperone activity.