Browsing by Author "Alvarez, James V"

The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of cytidine deaminases is one of the most common endogenous sources of single base substitution mutations in human cancer. Accordingly, APOBEC enzymes represent a major source of intratumor genetic heterogeneity and have been associated with immunotherapy response in diverse cancer types. However, the consequences of APOBEC mutagenesis on tumor progression in vivo are not well understood. To address this, I developed several murine tumor models with inducible APOBEC3B expression and studied the contribution of APOBEC activity to tumor evolution and immunogenicity. First, I explored the effects of APOBEC activity on tumor relapse using a murine model of mammary tumor recurrence. APOBEC activity led to a significant acceleration in tumor recurrence following the strong selective pressure of oncogenic driver signaling loss. Recurrent APOBEC tumors had undifferentiated histological features and large, irregularly shaped nuclei containing defects like micronuclei, multinucleation, and chromatin bridges. I found that recurrent APOBEC tumors amplified the therapy resistance-associated oncogene, c-Met, on circular extrachromosomal DNA, likely driving the proliferation of the recurrent cancer cells. Second, because APOBEC mutational signatures are enriched in the majority of HER2-positive breast cancer patients, I used a syngeneic HER2-driven mammary tumor model to study the effects of APOBEC activity on the tumor immune microenvironment. I found that APOBEC activity induced an antitumor adaptive immune response and CD4+ T cell-mediated tumor growth inhibition. While polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected by the immune system, suggesting that APOBEC-mediated genetic heterogeneity limits the antitumor adaptive immune response. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. Consistent with the observed immune infiltration in murine APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to checkpoint inhibition. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures and clonality as biomarkers predicting immunotherapy response in HER2-positive breast cancers. In conclusion, I’ve identified a novel role for APOBEC activity in generating chromosomal instability, consisting of mitotic errors, oncogene amplification, and extrachromosomal DNA formation to promote tumor recurrence. Moreover, APOBEC activity also stimulated an antitumor adaptive immune response and sensitized tumors to immunotherapy.

Tumor recurrence following initial treatment is the leading cause of death among breast cancer patients. Epigenetic mechanisms are critical for regulation of gene expression and to facilitate appropriate responses to environmental cues. However, it is increasingly appreciated that epigenetic dysregulation directly promotes therapeutic resistance and tumor progression. While genetic alterations have been shown to promote tumor progression, the contribution of non-genetic drivers of recurrence remains unexplored. In the current work, we utilized genetically engineered mouse models of breast cancer recurrence to evaluate the contribution of epigenetic plasticity to tumor recurrence and chemoresistance. First, we found that recurrent tumors undergo dramatic epigenetic and transcriptional reprogramming, partially through acquisition of an epithelial-to-mesenchymal transition (EMT). EMT promoted epigenetic silencing of tumor suppressor Par-4 through a unique, bivalent histone configuration. This bivalent configuration conferred plasticity to Par-4, and Par-4 silencing was reversed with epigenetic inhibitors of EHZ2 and HDAC. Further, Par-4 re-expression sensitized recurrent tumors to commonly utilized microtubule-targeting chemotherapeutics through altered cytoskeletal regulation. Second, we found that recurrent tumor epigenetic and transcriptional rewiring conferred sensitivity to G9a inhibitors. G9a inhibition promoted recurrent tumor cell necroptosis through demethylation of genes involved in a pro-inflammatory cytokine program. Further, knockout of G9a protein delayed the time until mammary tumors recurred in vivo. Collectively, our studies demonstrate that epigenetic dysregulation is a key feature of breast cancer progression, and pharmacologic strategies designed to target epigenetic enzymes underlying these processes may be of clinical value in the treatment of recurrent breast cancer.

Whole-genome duplication (WGD) generates polyploid cells possessing more than two copies of the genome. These events commonly occur during the evolution of human tumors across tissue types and mutational drivers, affecting an estimated 30-37% of all tumors. The frequency of WGD increases in advanced and metastatic tumors, and WGD is associated with poor prognosis in diverse tumor types, suggesting a functional role for polyploidy in tumor progression. Experimental evidence suggests that polyploidy has both tumor-promoting and suppressing effects. The polyploidization of a normally diploid cells often compromises genomic stability. In this way, WGD may be capable of promoting tumor formation, growth and progression, by facilitating the evolution of genetic heterogeneity on which selection can act. However, while some features of polyploidy can promote tumor growth, these features can also be countered by associated tumor suppressive qualities of polyploidization and associated cellular stresses. Chromosomal instability and resulting aneuploidy often have negative effects on cellular fitness; this can occur through the induction of proteotoxic stress, replication stress and delayed proliferation. Polyploidization can also be opposed by cell intrinsic and extrinsic pathways, including p53, the Hippo pathway and immunosurveillance. How these diverse and multifaceted features of polyploid cells work together to regulate tumor progression remains unclear.

Using a genetically engineered mouse model of HER2-driven breast cancer, we explored the prevalence and consequences of whole-genome duplication during tumor growth and recurrence. While primary tumors in this model are invariably diploid, nearly 40% of recurrent tumors undergo WGD. WGD in recurrent tumors was associated with increased chromosomal instability, decreased rates of proliferation and increased survival in stress conditions. The effects of WGD on tumor growth were dependent on tumor stage. Surprisingly, in recurrent tumor cells, WGD slowed tumor formation, tumor growth rate and opposed the process of recurrence, while WGD promoted the growth of primary tumors. Our findings highlight the importance of identifying conditions that promote the growth of polyploid tumors, including the cooperating genetic mutations that allow cells to overcome the barriers to WGD tumor cell growth and proliferation.

While our results revealed fitness disadvantages for recurrent polyploid tumor cells, the paradox remains that WGD is common in cancer cells despite this, suggesting that cells must evolve ways to overcome barriers to tumorigenesis. These findings suggest that a polyploid cancer cell may be delicately balanced, relying on certain pathways or processes to compensate for its cellular deficiencies more than their diploid counterparts. Ploidy-specific lethality describes the phenomenon in which inhibiting the activity or expression of a specific protein results in death of polyploid cells but not their diploid counterparts. To interrogate this idea, we next employed our models of recurrent polyploid cells to explore the impact of polyploidization on gene expression and signaling dependencies. Using RNA sequencing we uncovered that tetraploid cells exhibited decreased expression of genes of the cGAS-STING pathway. We performed two loss-of-function CRISPR screens against the kinome, one in vitro and one in vivo, to identify ploidy-specific lethal genes. The in vivo screen revealed candidates for ploidy-specific lethal genes including Srpk1, Mark4 and Ryk. Together these results demonstrated that polyploid recurrent tumor cells exhibit unique gene expression patterns that may reflect selection pressure of the immune system and may rely on unique survival mechanisms in vivo.

The survival and eventual recurrence of dormant residual tumor cells following therapy is a leading cause of death in many tumor types. The metabolic properties of dormant residual tumor cells, which are thought to be quiescent or slowly proliferating, are likely distinct from those of rapidly growing tumors. However, it is not known whether alterations in cellular metabolism directly regulate the survival of dormant cells or their reactivation to form recurrent tumors. To address this, we used a conditional mouse model of Her2-driven breast cancer to study metabolic adaptations following Her2 inhibition, during dormancy, and after tumor recurrence. First, we found that Her2 downregulation caused widespread changes in cellular metabolism, culminating in oxidative stress. Tumor cells adapted to this metabolic stress by upregulation of the antioxidant transcription factor, NRF2. Constitutive NRF2 expression persisted during dormancy and in recurrent tumors. Constitutive activation of NRF2 accelerated recurrence, while suppression of NRF2 impaired recurrent tumor growth. These results are supported by clinical data showing that the NRF2 transcriptional program is activated in recurrent breast tumors, and that NRF2 is associated with poor prognosis in patients with breast cancer. Mechanistically, NRF2 signaling in recurrent tumors induced metabolic reprogramming to re-establish redox homeostasis and upregulate de novo nucleotide synthesis. An in vivo CRISPR screen identified genes in the redox and nucleotide pathways as the essential downstream mediators of NRF2 in recurrent tumors. The NRF2-driven metabolic state rendered recurrent tumor cells sensitive to glutaminase inhibition, and glutaminase inhibition prevented reactivation of dormant tumor cells, suggesting that NRF2-high dormant and recurrent tumors can be therapeutically targeted. Together, these data provide evidence that NRF2-driven metabolic reprogramming promotes the recurrence of dormant breast cancer. Second, we found that the metabolic enzyme Bcat1 is upregulated in recurrent tumors as a result of epithelial-to-mesenchymal transition. We found that Bcat1 knockout impaired recurrent tumor growth, demonstrating its potential as a therapeutic target for recurrent tumors.

Cellular senescence is a unique cell fate characterized by stable cell cycle arrest and the extensive production and secretion of various cytokines, chemokines, proteases, and growth factors, a phenomenon known as the senescence-associated secretory phenotype (SASP). Although secreted factors are known to have important biological effects on both senescent and non-senescent cells in the contexts of normal aging and disease, the precise molecular mechanisms responsible for generating a SASP in response to senescent stimuli have remained largely obscure. To identify the major initiator, we used an unbiased profiling strategy and discovered a multi-ligand scavenger receptor CD36 is rapidly upregulated in multiple cell types in response to replicative, oncogenic and chemical senescent stimuli. Moreover, ectopic CD36 expression in dividing mammalian cells is sufficient to initiate the production of a large subset of known components of the SASP via activation of the canonical Src-NFκB pathway, resulting in the subsequent onset of a full senescent state. The CD36-mediated secretome is further shown to be ligand-dependent, as fibroblast cultures lacking the CD36 ligand amyloid beta (Aβ) are unresponsive to CD36 upregulation but can be driven to senesce by the addition of exogenous ligand. Finally, loss-of-function experiments revealed a strict requirement for CD36 in secretory molecule production during conventional senescence reprogramming. These results uncover the Aβ-CD36-NFκB signaling axis as an important regulator of the senescent cell fate via induction of the SASP.

To further explore the possible implication of Aβ-CD36-NFκB-SASP signaling, we found that the CD36 expression is significantly down-regulated in the context of lung malignant tissues, specifically in cancer cells. Subsequent explorations revealed CD36 as a strong tumor suppressor by secreting pro-inflammatory cytokines and recruiting cytotoxic T. For the CD36 ligand - Aβ, we observed a major accumulation in the tumor region which might serve as the tumor-suppressing signaling initiation cue once CD36 is introduced. The findings indicate a possible tumor suppressive signaling lead by Aβ-CD36.

Taken together, we discovered a novel signaling of Aβ-CD36-NFκB in regulating SASP during the process of lung aging and the progression of lung malignancy.

Over half of breast cancer related deaths are due to recurrence five or more years after initial diagnosis and treatment. This latency suggests that a population of residual tumor cells can survive treatment and persist in a dormant state for many years. The role of the microenvironment in regulating the survival and proliferation of residual cells following therapy remains unexplored. Using a conditional mouse model for Her2-driven breast cancer, we identify interactions between residual tumor cells and their microenvironment as critical for promoting tumor recurrence. Her2 downregulation leads to an inflammatory program driven by TNFα/NFκB signaling, which promotes immune cell infiltration in regressing and residual tumors. The cytokine CCL5 is elevated following Her2 downregulation and remains high in residual tumors. CCL5 promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. Blocking this TNFα-CCL5-macrophage axis may be efficacious in preventing breast cancer recurrence.

In addition, it remains unclear how the clonal composition of tumors changes during tumor relapse. We used cellular barcoding to directly monitor clonal dynamics during tumor recurrence in a genetically engineered mouse model. We found that the clonal diversity of tumors progressively decreased during tumor regression, residual disease, and recurrence. Only a fraction of subclones survived oncogene withdrawal and persisted in residual tumors. The minimal residual disease phase itself was accompanied by a continued attrition of clones, suggesting an ongoing process of selection during dormancy. The reactivation of dormant residual cells into recurrent tumors followed several distinct evolutionary routes. Approximately half of the recurrent tumors exhibited a striking clonal dominance in which one or two subclones comprised the vast majority of the tumor. The majority of these clonal recurrent tumors exhibited evidence of de novo acquisition of Met amplification, and were sensitive to small-molecule Met inhibitors. A second group of recurrent tumors exhibited marked polyclonality, with thousands of subclones and a clonal architecture very similar to primary tumors. These polyclonal recurrent tumors were not sensitive to Met inhibitors, but were instead dependent upon an autocrine IL-6 – Stat3 pathway. These results suggest that the survival and reactivation of dormant tumors can proceed via multiple independent routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

Advances in anti-cancer therapies, including chemotherapies, targeted therapies, and immuno-therapies, have drastically improved patient outcomes over the last few decades. However, tumor recurrence and acquired drug resistance continue to be detrimental for cancer patients, accounting for the majority of cancer-related deaths. Drug resistance is a common phenomenon that occurs when pharmaceutical agents are tolerated by and no longer effective against their target. In the case of cancer, acquired drug resistance is when cancer cells are able to survive, actively proliferate, and migrate to distant sites in the presence of anti-cancer agents. As such, it is imperative that we understand the mechanisms that lead to acquired drug resistance and tumor recurrence and leverage this newfound understanding to improve treatment options and, ultimately, patient outcomes.

Recurrent tumors and drug resistant cancer cells arise from drug-tolerant persister cells (DTPs), a population of cells that is able to withstand and adapt to cancer treatment. APOBEC3 and NRF2 are proteins that are largely absent and inactive in primary non-small cell lung cancer and HER2+ breast cancer, respectively. Interestingly, both proteins are found in recurrent disease, specifically after targeted therapy. This suggests that the upregulation of APOBEC3 and NRF2 is an adaptive response to anti-cancer treatment. Further, this suggests that APOBEC3 and NRF2 are essential for drug-tolerant persister cell survival and subsequent recurrence. Understanding how these proteins are induced, regulated, and what their respective roles are in tumor evolution can provide insight into therapeutic potentials in both residual and recurrent disease. First, I explored the regulation and function of APOBEC mutagenesis during acquired resistance to epidermal growth factor receptor (EGFR) inhibitors in two non-small cell lung cancer cell lines, PC9 and HCC827. I assess the effects of EGFR inhibition on APOBEC3 expression and activity and find that this induces APOBEC3 activity. Moreover, I evaluate how sustained APOBEC3 activity promotes evolution of drug resistance in DTPs. Finally, I examine what mechanisms might play a role in acquired drug resistance when APOBEC3 is highly active. I show that while APOBEC activity does not accelerate acquired therapy resistance, A3B expression alters the evolutionary path that PC9 cells take to become gefitinib-resistant. Specifically, A3B expression is associated with the late acquisition of T790M mutations during the DTP state, supporting a model where induction of APOBEC activity promotes DTP survival, thereby facilitating the on-going evolution of drug-tolerant persister cells. Of note, I find that APOBEC3 activity is associated with squamous cell transdifferentiation in PC9 cells, suggesting that p63 and its target genes could be future biomarkers and therapeutic targets.

Second, I investigated the regulation of the transcription factor NRF2 in recurrent breast cancer cells. I assess KEAP1-mediated regulation of NRF2 and find that while KEAP1 knockout in primary and recurrent cells caused an increase in NRF2 and its target genes, NRF2 remained more elevated in recurrent cells, indicating that increased NRF2 levels and transcriptional activity in these cells are independent of KEAP1. Next, I looked at post-translational modifications on NRF2 to determine if this may be the cause of the differential NRF2 levels in primary and recurrent cells. I found that NRF2 in recurrent cells had higher levels of phospho-Ser364, potentially affecting NRF2’s stability. Finally, I evaluated regulation of NRF2 by Akt and GSK-3β. I found that inhibition of Akt had no effect on NRF2 levels. In contrast to this, I found that GSK-3β activity is inversely correlated with NRF2 levels, suggesting that low levels of GSK-3β activity is partially responsible for NRF2 stabilization in recurrent tumor cells.

In conclusion, I have modeled two adaptive mechanisms for tumor recurrence and acquired drug resistance in two different cancer types. I elucidated the mechanisms by which APOBEC3B and NRF2 are able to promote cancer cell evolution in drug-tolerant persister cells that eventually give rise to recurrences.