2026 Theses Doctoral

The Role of Aneuploidy in Tumor Development and Outcome

Mallick, Samyukta E.

Over 90% of solid tumors in humans feature high rates of aneuploidy, the gain or loss of entire chromosomes or chromosome arms. Despite being detected as a tumor-associated genomic alteration over a century ago, the effects of specific aneuploidy events in cancer biology remain largely uncharacterized. Although aneuploidy events were originally described as a byproduct of the unconstrained proliferation, suppression of DNA damage repair pathways, and resulting chromosomal instability characteristic of neoplastic growth, recent work has described aneuploidy events preceding tumor formation, suggesting a key role in early tumorigenesis. Moreover, analyses have revealed recurring patterns of aneuploidy alterations acquired by specific tumor types, suggesting that these genomic alterations may have targeted roles in tumor development. Here, we leverage both correlative and mechanistic studies to investigate how these recurrent aneuploidy events affect cancer cells, the tumor microenvironment, and patient outcomes. By expanding our analyses of patient data to these widescale genomic alterations, we may gain a better understanding of pathological mechanisms, as well as help stratify patients and develop more effective treatment plans.

Aneuploidy events constitute simultaneous copy number alteration (CNA) of hundreds of genes and trigger compensatory mechanisms, which together complicate our mechanistic understanding of these events. Moreover, because murine and human chromosomes do not have synteny, studies in animal models fail to recapitulate human biology. Previous work in our lab has developed a CRISPR-Cas9 genome editing system to model aneuploidy events in vitro by replacing an entire chromosome arm with an artificial telomere. Using this system, I first seek to understand the effects of chromosome arm 3p deletion on tumorigenesis in head and neck squamous cell carcinoma (HNSC), the sixth most common cancer in the world. HNSC tumorigenesis is driven either by carcinogen exposure or infection with human papilloma virus (HPV). Recent analyses have uncovered unique patters of aneuploidy events during progression of viral- or carcinogen-driven HSNC. Notably, 3p deletion occurs much more frequently in HPV– tumors compared to HPV+ tumors, suggesting that HPV infection and 3p deletion may serve redundant purposes during tumor development. Here, we engineered isogenic gingival basal cells with HPV E6/E7 overexpression and lung basal cells with 3p deletion to investigate the effects of these alterations on cell proliferation, protein localization, and differentiation. We found that both HPV E6/E7 and hemizygous 3p deletion trigger increased expression of proliferation signatures, decreased rates of squamous differentiation, and downregulation of proteins necessary for cell-cell junctions and cellular polarity.

These mechanistic insights into HPV-associated 3p deletion prompted a broader investigation of recurrent chromosome arm-level aneuploidies across tumor types. We leveraged the Cancer Genome Atlas (TCGA) to investigate the role of various aneuploidy events, namely 3p and 19p loss due to their high frequency, in different tumor types, identifying arm level correlations with hallmark pathway expression across tumor types. As immunotherapy, namely immune checkpoint blockade (ICB), has become a first-line therapy for many cancer types, we sought to explore the associations of aneuploidy with immune signaling and immune cell infiltration in different tumor types. Here we see inverse associations of immune signaling with 3p deletion between SCC and KIRC tumors that appear to be driven by changes in the TME (tumor microenvironment), rather than breakpoint differences. Conversely, we see differences in immune signaling associated with 19p deletion between LUAD and LUSC that cannot be explained by immune infiltrate, suggesting that cell- intrinsic cytokine signaling may be driving immune signature differences. This is particularly important because despite their vast differences, LUAD and LUSC cases are often treated similarly since they both fall under NSCLC.

Finally, we investigate the implications of copy number alterations (CNA) of STK11 and KEAP1, tumor suppressors that regulate AMPK and mTOR signaling, and NRF2 activity, respectively. Mutations in these genes are associated with poor survival in non-small cell lung cancer (NSCLC), and chromosome 19p (chr19p), on which they reside. Given the clinical importance of immune checkpoint blockade in NSCLC, we next examined whether chr19p copy number status associates with outcomes in PD-(L)1i (PD-1/PD-L1 inhibitor)-treated patients. In particular, we sought to characterize these alterations with respect to NSCLC histology and clinical outcome via overall survival (OS). We analyzed mutation, copy number, and survival data from patients across three NSCLC cohorts consisting of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients: TCGA, AACR GENIE, and the Stand Up to Cancer-Mark Foundation. Whereas STK11 and KEAP1 mutations occur in 1% of LUSC and 10% of LUAD, hemizygous deletion by focal loss or chr19p arm deletion occurs in 20-50% of both NSCLC subtypes. Subtype-specific analysis demonstrated that patients with LUSC showed better survival with chr19p gain (HR 0.63 p = 0.039). Patients treated with PD-(L)1i immunotherapy showed association with worse outcome, regardless of histology, with chr19p deletion (LUAD HR 1.45, p = 0.034; LUSC HR 4.09, p = 0.0021), suggesting that 19p gene copy number alterations define clinically relevant subsets of not just LUAD, but LUSC as well. Our study highlights that 19p copy number status should be studied prospectively in patients with NSCLC to further establish the clinical relevance of these common alterations as well as their biological implications.

Together, although a complete understanding of the effects of aneuploidy events still eludes the field, our results indicate potential mechanisms by which aneuploidy contributes to tumor development and progression, uncover site-specific effects of specific aneuploidy events which will direct future mechanistic studies, and identify utility in tracking these aneuploidy events to stratify patients and better predict outcomes.