Vaidehi Krishnan

Disruption of Runx1 and Runx3 leads to bone marrow failure and leukemia predisposition due to transcriptional and DNA repair defects.

The RUNX genes encode transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here, we show that Runx1;Runx3 double-knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes such as Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independently of CBFβ, suggesting a nontranscriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes the development of leukemias and other cancers.

Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression

Significance The Runt-related transcription factors (RUNX) are critical regulators of development. Mutation or dysregulation of RUNX genes have been associated with diverse cancer types. Phosphorylation of the strictly conserved threonine 173 (T173) residue within the Runt domain of RUNX3 disrupts DNA binding activity, which facilitates the recruitment of RUNX proteins to mitotic structures. Moreover, RUNX3 deficiency is associated with delayed mitotic entry. The tight conservation of T173 and its flanking residues from unicellular organism to human, and the fact that mitosis is indispensable to all metazoans, strongly indicate that T173 phosphorylation is fundamental to the role of RUNX in these divergent organisms. Cancer-associated mutation T173I further corroborates the critical role of RUNX phosphorylation in mitosis. The Runt-related transcription factors (RUNX) are master regulators of development and major players in tumorigenesis. Interestingly, unlike most transcription factors, RUNX proteins are detected on the mitotic chromatin and apparatus, suggesting that they are functionally active in mitosis. Here, we identify key sites of RUNX phosphorylation in mitosis. We show that the phosphorylation of threonine 173 (T173) residue within the Runt domain of RUNX3 disrupts RUNX DNA binding activity during mitotic entry to facilitate the recruitment of RUNX proteins to mitotic structures. Moreover, knockdown of RUNX3 delays mitotic entry. RUNX3 phosphorylation is therefore a regulatory mechanism for mitotic entry. Cancer-associated mutations of RUNX3 T173 and its equivalent in RUNX1 further corroborate the role of RUNX phosphorylation in regulating proper mitotic progression and genomic integrity.

ATM Expression Predicts Veliparib and Irinotecan Sensitivity in Gastric Cancer by Mediating P53-Independent Regulation of Cell Cycle and Apoptosis.

Identification of synthetically lethal cellular targets and synergistic drug combinations is important in cancer chemotherapy as they help to overcome treatment resistance and increase efficacy. The Ataxia Telangiectasia Mutated (ATM) kinase is a nuclear protein that plays a major role in the initiation of DNA repair signaling and cell-cycle check points during DNA damage. Although ATM was shown to be associated with poor prognosis in gastric cancer, its implications as a predictive biomarker for cancer chemotherapy remain unexplored. The present study evaluated ATM-induced synthetic lethality and its role in sensitization of gastric cancer cells to PARP and TOP1 inhibitors, veliparib (ABT-888) and irinotecan (CPT-11), respectively. ATM expression was detected in a panel of gastric cell lines, and the IC50 against each inhibitors was determined. The combinatorial effect of ABT-888 and CPT-11 in gastric cancer cells was also determined both in vitro and in vivo. ATM deficiency was found to be associated with enhanced sensitivity to ABT-888 and CPT-11 monotherapy, hence suggesting a mechanism of synthetic lethality. Cells with high ATM expression showed reduced sensitivity to monotherapy; however, they showed a higher therapeutic effect with ABT-888 and CPT-11 combinatorial therapy. Furthermore, ATM expression was shown to play a major role in cellular homeostasis by regulating cell-cycle progression and apoptosis in a P53-independent manner. The present study highlights the clinical utility of ATM expression as a predictive marker for sensitivity of gastric cancer cells to PARP and TOP1 inhibition and provides a deeper mechanistic insight into ATM-dependent regulation of cellular processes. Mol Cancer Ther; 15(12); 3087–96. ©2016 AACR.

TGFβ Promotes Genomic Instability after Loss of RUNX3

Studies of genomic instability have historically focused on intrinsic mechanisms rather than extrinsic mechanisms based in the tumor microenvironment (TME). TGFβ is the most abundantly secreted cytokine in the TME, where it imparts various aggressive characteristics including invasive migration, drug resistance, and epithelial-to-mesenchymal transition (EMT). Here we show that TGFβ also promotes genomic instability in the form of DNA double strand breaks (DSB) in cancer cells that lack the tumor suppressor gene RUNX3 Loss of RUNX3 resulted in transcriptional downregulation of the redox regulator heme oxygenase-1 (HO-1 or HMOX1). Consequently, elevated oxidative DNA damage disrupted genomic integrity and triggered cellular senescence, which was accompanied by tumor-promoting inflammatory cytokine expression and acquisition of the senescence-associated secretory phenotype (SASP). Recapitulating the above findings, tumors harboring a TGFβ gene expression signature and RUNX3 loss exhibited higher levels of genomic instability. In summary, RUNX3 creates an effective barrier against further TGFβ-dependent tumor progression by preventing genomic instability. These data suggest a novel cooperation between cancer cell-extrinsic TGFβ signaling and cancer cell-intrinsic RUNX3 inactivation as aggravating factors for genomic instability.Significance: RUNX3 inactivation in cancer removes an antioxidant barrier against DNA double strand breaks induced by TGFβ expressed in the tumor microenvironment. Cancer Res; 78(1); 88-102. ©2017 AACR.

The Fanconi Anemia Pathway of DNA Repair and Human Cancer

The accurate repair of DNA damage and the maintenance of genomic integrity is a funda‐ mental property of every cell. Amongst the different classes of DNA damaging agents, DNA interstrand crosslinks (ICLs) represent a class of DNA lesions wherein the two strands of DNA get cross-linked by covalent bonds. Unrepaired, such cross-linking will impede the progress of critical processes like DNA replication and transcription, resulting in a genomic instabilityassociated disorder called, Fanconi Anemia (FA).

HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells

Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24−/− mice. The results showed that the level of HP1α was significantly increased in Zmpste24−/− cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24−/− MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24−/− MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24−/− MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.

Aurora kinase and RUNX: Reaching beyond transcription.

The RUNX family of genes are well established as transcription factors essential for differentiation and development in higher organisms. The RUNX family is profoundly implicated in cancer: dysregulation of RUNX genes has been linked to initiation as well progression of diverse cancer types. RUNX proteins derived their name from the Runt domain, an evolutionarily conserved 128-amino acid region, which endows RUNX proteins with sequence-specific DNA binding. Interestingly, unlike many transcription factors in mitosis, RUNX proteins are found on key mitotic structures such as the centrosome, spindle and midbody – raising the intriguing possibility that RUNX proteins play active roles in mitosis, a cell cycle phase traditionally associated with major cessation of transcription. One of the most obvious changes to the RUNX proteins during mitosis is the massive phosphorylation of all 3 members of the human RUNX family. And yet, the underlying reason remains obscure. Recently, we found that 2 key residues at the N-terminus domain of RUNX3 – threonine 14 (T14) and threonine 173 (T173) – are critical for mitotic-specific hyperphosphorylation of RUNX3. The crystal structure of the Runt domain showed that the peptide comprising T173 and its flanking amino acid residues constitutes a key structure necessary for the Runt-DNA interaction; moreover, T173 contacts the phosphate backbone of the DNA double helix via polar interaction. Mitosis-specific phosphorylation of T173 regulates RUNX3 subcellular localization through inhibition of the DNA-binding function of the Runt domain – RUNX proteins are detached from the DNA and redistributed to the cytoplasm and mitotic structures such as the centrosome and midbody, which are necessary for spindle formation and cytokinesis, respectively. Aurora kinases are master regulators of mitosis with similar localization patterns as RUNX3. The identification of RUNX3 as a novel substrate of Aurora kinases, which induces T173 phosphorylation, therefore suggests non-canonical roles for RUNX3 at specific stages of mitosis. We showed that RUNX3 knockdown results in delayed mitotic entry while RUNX3 phosphorylation is a likely regulatory element for mitotic entry. The strong conservation of T173 and its flanking residues in divergent organisms, from the unicellular holozoan Capsaspora owczarzaki to human, indicates that T173 phosphorylation evolved to regulate the primordial role of RUNX and perhaps, tailor it to mitosis. Mutation of the T173 residue and equivalents – all to isoleucine – were discovered in all RUNX family members in human diseases. In particular, RUNX3 (T173I) and RUNX1 (equivalent, T196I) mutations were cancer associated (http://cancer.sanger.ac. uk). Unlike wild-type RUNX3, T173 mutants promoted colony formation on soft agar, suggesting that mutation or phosphorylation of T173 is associated with loss of growth inhibition. Our work suggests that T173 phosphorylation normally acts to regulate mitosis but with the accompanying caveat: the risk of cancer formation if T173 phosphorylation is not tightly confined to mitosis. Since the tumor suppressor property of RUNX3 mainly resides in its ability to regulate transcription of genes that induce cell cycle arrest, apoptosis and senescence, the frequent upregulation of Aurora kinases in cancer could reasonably be expected to attenuate RUNX3 transcription activity, and therefore its tumor suppressor function, during non-mitotic phases. We had earlier reported an acetylation site at lysine 171 (K171), which is important for complex formation between RUNX3 and bromodomain-containing protein BRD2 in a KRasdependent manner. 6 The RUNX3-BRD2 complex mediates transcription of growth inhibitory and tumor suppressor genes p21 and p14 respectively to protect cells from oncogenic signals. Aurora kinase B preferentially phosphorylates threonine/serine residues with upstream basic residues. K171, at the –2 position, conforms to an Aurora kinase B target motif (Fig. 1). Acetylation of K171 might therefore interfere with phosphorylation of T173. It is conceivable that the conservation of the phospho-site T173 and acetyl-site K171 is due to selective pressure for cross-check between modifications – as a safeguard against tumorigenesis. Finally, our work reinforced the exciting notion that all RUNX proteins possess non-transcriptional roles. We propose that RUNX proteins have transcriptional and non-transcriptional roles that function in a complementary manner to maintain cell identity. Earlier, we reported that RUNX1 and RUNX3 are integral

DNA Repair, Human Diseases and Aging

One of the most fundamental functions of a cell is the transmission of genetic information to the next generation, with high fidelity. On the face of it, this seems a challenging task given that cells in the human body are constantly exposed to thousands of DNA lesions every day both from endogenous and exogenous sources. Yet, for long periods of time, the DNA sequences are one of the most invariable and stable components of a cell, a task achieved by an arsenal of DNA damage detection and repair machinery that detects and fixes DNA lesions with high fidelity at each and every round of cell division. In this chapter, we describe how normal cells cope with DNA damage and the manner in which a defective DNA damage response can lead to human disease and aging.