Yasuhiko Kawakami

MYC-driven (MYC ⁺ ) cancers are highly aggressive and often fatal. MYC dysregulation is a key event in these cancers, yet MYC overexpression alone is often insufficient to initiate or sustain tumorigenesis. Plasmocytoma variant translocation 1 ( PVT1 ), a long noncoding RNA (lncRNA) adjacent to MYC on chromosome 8q24, is frequently co-amplified with MYC in many of these cancers. Our prior work showed that PVT1 potentiates MYC function, although the underlying mechanism has remained unclear. Here we show that, in addition to amplification with MYC , genomic rearrangements at PVT1 frequently involve unbalanced translocations that asymmetrically enrich 5′- PVT1 while depleting 3′- PVT1 . The retained 5′- PVT1 region generates a circular RNA, CircPVT1 , that encodes a novel protein we have named Firefox (FFX). We show that FFX is essential for MYC-mediated oncogenic signaling, as its depletion markedly reduces MYC protein abundance and transcriptional output. Mechanistically, FFX stimulates AKT–mTORC1 signaling and enhances cap-dependent translational and biosynthetic capacity, thereby establishing a self-reinforcing oncogenic circuit that amplifies MYC activity. Inducible depletion of FFX in vivo significantly impairs tumor growth in MYC ⁺ xenograft models. These findings define FFX as a critical effector within the MYC - PVT1 locus and reveal a therapeutically actionable vulnerability in MYC ⁺ cancers.

M⁶A RNA methylation is essential for many aspects of mammalian development but its roles in chondrogenesis remain largely unknown. Here, we show that m⁶A is necessary for chondrogenesis and limb morphogenesis using limb progenitor-specific knockout mice of Mettl14, an essential subunit in the m⁶A methyltransferase complex. The knockout disrupts cartilage anlagen formation in limb buds with 11 downregulated proteins known to dysregulate chondrogenesis and shorten limb skeletons upon mutation in mice and humans. Further studies show a gene regulatory hierarchy among the 11 proteins. m⁶A stabilizes the transcript and increases the protein level of GDF5, a BMP family member. This activates the chondrogenic transcription factor genes Runx2 and Runx3, whose mRNAs are also stabilized by m⁶A. They promote the transcription of six collagen genes and two other chondrogenic genes, Ddrgk1 and Pbxip1. Thus, this study uncovers an m⁶A-based cascade essential for chondrogenesis during limb skeletal development.

MYC-driven (MYC+) cancers are aggressive and often fatal. MYC dysregulation is a key event in these cancers, but overexpression of MYC alone is not always enough to cause cancer. Plasmocytoma Variant Translocation 1 ( PVT1 ), a long non-coding RNA (lncRNA) adjacent to MYC on chromosome 8 is a rearrangement hotspot in many MYC+ cancers. In addition to being co-amplified with MYC, the genomic rearrangement at PVT1 involves translocation, which has had obscure functional consequences. We report that translocation at the PVT1 locus cause asymmetric enrichment of 5’-PVT1 and loss of 3’-PVT1. Despite being classified as a non-coding RNA, the retained 5’ region of PVT1 generates a circular RNA (CircPVT1) that codes for the novel peptide we call Firefox (FFX). FFX augments AKT signaling and synergistically activates MYC and mTORC1 in these cells. Further, the 3’ end of PVT1, which is lost during the translocation, codes for a tumor-suppressing micropeptide we named as Honeybadger (HNB). We demonstrate that HNB interacts with KRAS and disrupts the activation of KRAS effectors. Loss of HNB leads to activation of RAS/MAPK signaling pathway, and enhances MYC stability by promoting phosphorylation of MYC at Ser ⁶² . These findings identify PVT1 as a critical node that synchronizes MYC, AKT, and RAS-MAPK activities in cancer. Our study thus identifies a key mechanism by which rearrangements at the PVT1 locus activate additional oncogenic pathways that synergize with MYC to exacerbate the aggressiveness of MYC+ cancers. This newfound understanding explains the poor prognosis associated with MYC+ cancers and offers potential therapeutic targets that could be leveraged in treatment strategies for these cancers.

The mammalian heart loses almost all its regenerative potential in the first week of life due to the cessation of the ability of cardiomyocytes to proliferate. In recent years, a number of regulators of cardiomyocyte proliferation have been identified. Despite this, a clear understanding of the regulatory pathways that control cardiomyocyte proliferation and cardiac regeneration is lacking, and there are likely additional regulators to be discovered. Here, we performed a genome-wide screen on fetal murine cardiomyocytes to identify potential novel regulators of cardiomyocyte proliferation. Endoglin was identified as an inhibitor of cardiomyocyte proliferation in vitro. Endoglin knock-down resulted in enhanced DNA synthesis, cardiomyocyte mitosis and cytokinesis in mouse, rat and human cardiomyocytes. Using gene-targeted mice, we confirmed myocardial Endoglin to be important in cardiomyocyte proliferation and cardiac regeneration using gene-targeted mice. Mechanistically, we show that Smad signaling is required for the endoglin-mediated anti-proliferative effects. Our results identify the TGF-β coreceptor Endoglin as a regulator of cardiac regeneration and cardiomyocyte proliferation. Summary: High-content function screening is used to identify a novel inhibitor of cardiomyocyte proliferation which can promote mammalian cardiac regeneration.

Cardiomyocyte (CM) proliferation and maturation are highly linked processes, however, the extent to which these processes are controlled by a single signaling axis is unclear. Here, we show the previously undescribed role of Hedgehog (HH)-GLI2-CKS1B cascade in regulation of the toggle between CM proliferation and maturation. Here we show downregulation of GLI-signaling in adult human CM, adult murine CM, and in late-stage hiPSC-CM leading to their maturation. In early-stage hiPSC-CM, inhibition of HH- or GLI-proteins enhanced CM maturation with increased maturation indices, increased calcium handling, and transcriptome. Mechanistically, we identified CKS1B, as a new effector of GLI2 in CMs. GLI2 binds the CKS1B promoter to regulate its expression. CKS1B overexpression in late-stage hiPSC-CMs led to increased proliferation with loss of maturation in CMs. Next, analysis of datasets of patients with heart disease showed a significant enrichment of GLI2-signaling in patients with ischemic heart failure (HF) or dilated-cardiomyopathy (DCM) disease, indicating operational GLI2-signaling in the stressed heart. Thus, the Hh-GLI2-CKS1B axis regulates the proliferation-maturation transition and provides targets to enhance cardiac tissue engineering and regenerative therapies.

The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.

Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which trigger molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5) and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing one allele Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation.