OS is a genetically complex and heterogeneous tumor characterized by chromosomal instability and genetic alterations that lead to aneuploidy and increased tumor aggressiveness. The high rates of chromosomal rearrangements in OS include structural chromosomal abnormalities, such as translocations, deletions, amplifications, and chromothripsis, an extreme form of chromosomal instability [12]. Chromothripsis is associated with increased genomic instability and tumor progression, and it frequently occurs in highly aggressive tumors, including OS. Although the exact cause of chromothripsis and its role in tumorigenesis remain unclear, recent genomic studies have revealed that it is context-dependent and occurs at an overall incidence of between 2% and 3% in pan-cancer samples but over 77% in OS and 100% in liposarcoma [13]. Some potential mechanisms underlying chromothripsis are emerging, including the generation of DNA breaks and rejoining of the DNA fragments, generation of micronuclei, premature chromosome condensation, breakage–fusion–bridge cycle and telomere dysfunction, and ionizing radiation [14]. While the exact mechanistic cause of chromothripsis is still undefined, Crasta et al. identified the micronuclei, having many features of primary nuclei, formed from the acentric fragments of chromosomes, which produce DNA damage behind the chromothripsis [15]. Zhang et al., by using a combination of live cell imaging and single-cell genome sequencing, demonstrated that micronucleus formation could indeed generate a spectrum of genomic rearrangements, which recapitulate the features of chromothripsis [16]. Gong et al. described how Ran GTPase-activating protein 1 (RanGAP1) is commonly reduced or inactivated in human OS, leading to a high probability of chromothripsis, which drives tumorigenesis through its direct effects on the spindle-assembly checkpoint and decatenation and secondary effects on DNA damage surveillance [17].

Inactivation of tumor suppressor genes such as TP53, RB1, ATRX, and DLG2 is frequently observed in OS, which is thought to be involved in OS tumorigenesis [18]. The TP53 gene is OS’s most frequently dysregulated gene [18]. Whole-genome DNA sequencing from OS tumor samples demonstrates multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). Kataegis is a single nucleotide variation (SNV) detected in 50% of OS tumors. Chen et al. identified p53 pathway lesions in all OS patients, while it was translocated in around 50% of the patients to the first intron of the TP53 gene, leading to gene inactivation. This mechanism of TP53 gene inactivation is unique to OS among pediatric cancers [18]. The p53 protein is a tumor suppressor protein involved in DNA damage recognition that induces apoptosis, cellular quiescence, or senescence. Another tumor suppressor gene frequently inactivated in OS is RB1, located at chromosome 13q14.2 [19]. RB1 encodes the tumor suppressor protein pRB, which is vital for preventing cell cycle progression. ATRX is an important tumor suppressor in OS, and it is a part of a multiprotein complex that regulates chromatin remodeling, nucleosome assembly, and telomere maintenance. Furthermore, a recent report noted that loss of ATRX promotes OS tumor through increased NF-κB signaling and integrin binding [20]. DLG2 is a tumor suppressor gene, and its copy number loss occurs in 42% of human and 56% of canine OS [21]. Deletion of Dlg2 in a mouse model led to the acceleration of OS development [21].

Hyperactivation of tumor-promoting genes such as MYC and MDM2 is associated with OS tumorigenesis. The gain of the 8q24 chromosomal locus, which harbors the oncogene MYC, has been reported in several OS patients [22,23]. MYC is involved in cell cycle regulation, protein biogenesis, metabolism, signal transduction, transcription, and translation [24,25]. A recently generated Myc knock-in genetically engineered mouse model of an OS tumor not only identified intrinsic Myc-mediated mechanisms of OS tumorigenesis but also identified a novel molecular mechanism through which Myc regulates the profile and function of the OS immune landscape [26,27,28]. The oncoprotein MDM2 is a p53 inhibitor, which promotes p53 degradation and downregulates its transcription. Amplification of MDM2 (chromosome 12q15) is more frequent in OS metastasis and recurrence.

Epigenetic modifications, including DNA methylation, histone acetylation, and methylation, are critical in the pathogenesis of several cancers, including OS [29]. The level of histone H3 lysine trimethylation was reported to be lower in human OS tissue and cell lines compared with normal bone tissue and osteoblast cells. Enhancement of H3 methylation after treatment with the histone lysine demethylase inhibitor 5-carboxy-8-hydroxyquinoline (IOX-1) showed inhibition of OS migratory and invasive capabilities. Enhanced histone H3 lysine trimethylation levels sensitized cisplatin against the cisplatin-resistant (MG63-CR) cells [30]. Previous studies showed enhancement of the expression of the lysine-specific demethylases KDM1A, KDM2B, KDM4A, KDM6A, KDM6B in OS progression [31,32,33,34]. Recently, Twenhafel et al. provided a comprehensive review of recent advances in the epigenetics of OS and highlighted the clinical benefits in the field of OS research [35]. Morrow et al. highlighted the genetic and epigenetic defects in OS and emphasized the role of epigenetic dysregulation in tumor suppression and oncogene regulation [6].