Advancing Cancer Research with Next-Generation Sequencing Technologies

The application of next-generation sequencing (NGS) in cancer research has been instrumental in identifying genes related to cancer, improving our insight into tumor diversity, and uncovering mutations that lead to tumorigenesis. In a recent Bench Tip webinar, three presenters discussed their innovative cancer research highlighting the contributions of this influential technology. This article reviews their presentations while underscoring their significance and impact.

Analyzing drug response in MDS patients

Juan Jose Rodriguez-Sevilla, M.D., Ph.D., a Postdoctoral Fellow at the University of Texas MD Anderson Cancer Center, shared his work on the challenges of treating myelodysplastic syndromes (MDS), which encompass a range of disorders involving hematopoietic stem cells (HSCs). With around 50% of MDS patients not responding to standard hypomethylating agents and a high risk of progression to acute myeloid leukemia (AML),¹ Rodriguez-Sevilla's research focused on using various sequencing techniques to uncover treatment-resistance mechanisms in MDS.

In a previous study, his group analyzed over 400 primary MDS samples and identified two cell differentiation patterns: common myeloid progenitor (CMP) and granulocytic-monocytic progenitor (GMP).² They investigated whether the patterns from these hematopoietic stem and progenitor cells (HSPCs) could predict a response to a promising drug called venetoclax. The group observed that CMP pattern patients relied on BCL2-mediated anti-apoptotic pathways for survival, while GMP pattern patients depended on TNF-induced NF-κB signaling pathways.

The study further revealed that venetoclax targets “CMP pattern” HSCs. In a cohort of 21 MDS patients, CMP pattern patients responded quicker and had longer relapse-free survival. However, some CMP patients shifted to a GMP pattern, particularly noted in two patients with trisomy eight (+8). Sequencing data showed one patient developed a STAG2-mutant clone with additional mutations, while the other acquired RUNX1 mutations, suggesting a switch in oncogenic dependency from BCL2 to NF-κB.

The team also examined the role of trisomy eight in venetoclax resistance. While trisomy eight itself wasn't significantly associated with either CMP or GMP patterns, it was linked to STAG2 mutations, suggesting it may predispose CMP patients to develop specific mutations under venetoclax treatment, leading to resistance. To understand this process at a transcriptional level, single-cell RNA sequencing (scRNA-seq) was performed for the first two patients who showed this switch. The analysis showed an enrichment in the TNF-alpha signaling pathway during disease progression, supporting the hypothesis of an oncogenic shift.

Rodriguez-Sevilla proposed a model where CMP-pattern MDS patients treated with hypomethylating agents and venetoclax might switch to a GMP pattern due to new or expanded STAG2 or RUNX1 mutations, altering their oncogenic reliance. This model indicates that patients no longer respond to venetoclax as they do not rely on BCL2 for survival. In his conclusion, Rodriguez-Sevilla highlighted immunophenotypic switching as a new mechanism of venetoclax relapse in CMP-pattern MDS patients, underscoring the value of mutational monitoring to predict relapse and guide treatment adjustments.

Investigating the role of GATA3 in breast cancer

Motoki Takaku, Ph.D., an Assistant Professor from the University of North Dakota, presented his research on the roles of chromatin and GATA3 in breast cancer. He began by emphasizing the significance of chromatin in cellular reprogramming and differentiation, particularly during tumor progression. Central to this process are pioneer factors, a group of transcription factors that can interact with nucleosomal DNA and lead to local chromatin remodeling and activation of gene expression.

Takaku went on to describe GATA3's function as a pioneer transcription factor in the context of breast cancer. He detailed its significance, particularly its ability to reprogram certain aggressive and metastatic breast cancer cells into less aggressive epithelial cells.³ Previous research has also revealed that higher GATA3 expression is linked to better prognosis in breast cancer patients.⁴ However, the precise function of GATA3 and the impact of its frequent mutations in breast cancer cells remain unclear.

To explore this, Takaku’s team conducted investigations using a mutant cell line, focusing on the effects of GATA3 mutations. They observed that certain mutations led to more aggressive phenotypes, suggesting a role of GATA3 in breast cancer reprogramming. Using another experimental model, they demonstrated that overexpression of GATA3 reprogrammed these cells into a less metastatic, more epithelial phenotype.

In their investigation of chromatin reprogramming, the group utilized a DOX-inducible system to track chromatin changes over time. Through ChIP-seq and ATAC-seq analyses they found that GATA3 binding increased chromatin accessibility at certain sites, while other GATA3-bound regions remained closed. This discovery contradicted their previous assumptions about GATA3 as a pioneer transmission factor, prompting a more detailed investigation into chromatin structure.

Further exploration led the team to refine the MNase-seq technique with an enrichment step to capture regions of interest.⁵ This improvement allowed for precise observation of nucleosome positioning. Their findings indicated that GATA3 binding either reduced nucleosome occupancy or caused minor nucleosome shifts at different sites. Interestingly, GATA3 was also found to colocalize with chromatin remodeling factors such as BRG1 and CHD4.

Knockdown experiments with CHD4 demonstrated significant changes in nucleosome structure and aberrant chromatin opening, indicating a critical role of CHD4 in regulating GATA3's function in breast cancer cells. Through pathway analysis, Takaku’s team discovered that CHD4's absence leads to chromatin remodeling and the activation of genes typically associated with brain and placental cells, both linked to GATA3 function.

Takaku summarized his research with a working model where GATA3 expression in mesenchymal cells initiates reprogramming to epithelial cells, potentially reducing the aggressiveness of breast cancer cells. However, in the absence of CHD4, GATA3 will start activating genes related to other cell contexts. The group is now focused on understanding the cooperative function between the GATA3 and the CHD4.

Single-cell profiling shows heterogeneity in liposarcoma progression

Veena Kochat, Ph,D., a Senior Research Scientist at the University of Texas MD Anderson Cancer Center, presented her findings on the progression of well-differentiated (WD) to de-differentiated (DD) liposarcoma. This type of soft tissue tumor can evolve from low-grade adipocytic tumors in its WD stage to more aggressive, high-grade forms in the DD stage.⁶

To investigate this progression, Kochat's team employed single-cell multiomic profiling, a combination of scRNA-seq and single-cell ATAC-seq (scATAC-seq). This approach revealed a high degree of heterogeneity in liposarcomas. They identified several distinct clusters of tumor, immune, and stromal cells, each with unique gene expression profiles. These clusters were classified into cell types such as preadipocytes, adipocyte progenitors, and mesenchymal progenitors based on known gene signatures.

For a better understanding of the cellular hierarchy and differentiation states within the tumors, the team utilized computational tools such as CytoTRACE and inferCNV. CytoTRACE, in particular, helped to categorize cells based on their “stemness”—their potential to differentiate into various cell types. Surprisingly, some well-differentiated tumor samples showed higher stemness scores compared to their de-differentiated counterparts, challenging traditional pathology-based tumor classification.

Another key finding was the identification of a preadipocyte cluster present in all samples, which expanded during the transition from WD to DD liposarcoma in most samples. The inferCNV analysis showed several changes in the cluster, such as amplifications in chromosomes 12 and 13, indicating its potential role as a driver of tumor progression.

Integration of RNA-seq and ATAC-seq data further revealed different potential gene regulatory networks driving the evolution of these tumors. These networks were characterized by specific transcription factors that were distinct for tumor clusters, indicating a shift in the way genes are regulated during the transition from WD to DD liposarcoma.

Pseudotime analysis provided insights into diverse cell state trajectories and heterogeneity in the de-differentiation process across patient samples. It showed two primary pathways, epigenetically regulated, that play a crucial role in this evolution. These pathways exhibited distinct enhancer activities, uniquely characteristic of either WD or DD states.

Further investigation uncovered a significant transition toward zinc finger transcription factors as the liposarcoma transformed. These factors are commonly linked to stemness, indicating a shift in cellular identity during the tumor's progression. This finding is in stark contrast to the adipogenic regulation observed in WD states and underscores the significance of the transcription factors CTCF and BORIS found in DD states.

Finally, Kochat proposed that epigenetic plasticity, especially at CTCF binding sites involved in chromatin looping and enhancer-promoter interactions, is crucial in driving the diverse trajectories of cell states during the de-differentiation process in liposarcoma. This plasticity leads to a variety of genetic expressions and interactions, contributing to the complexity and variability seen in these tumors.

References

1. Prébet, T., Gore, S. D., Esterni, B., Gardin, C., Itzykson, R., Thepot, S., Dreyfus, F., Rauzy, O. B., Recher, C., Adès, L., Quesnel, B., Beach, C. L., Fenaux, P., & Vey, N. (2011). Outcome of high-risk myelodysplastic syndrome after azacitidine treatment failure. Journal of clinical oncology, 29(24), 3322–3327. 

2. Ganan-Gomez, I., Yang, H., Ma, F., Montalban-Bravo, G., Thongon, N., Marchica, V., Richard-Carpentier, G., Chien, K., Manyam, G., Wang, F., Alfonso, A., Chen, S., Class, C., Kanagal-Shamanna, R., Ingram, J. P., Ogoti, Y., Rose, A., Loghavi, S., Lockyer, P., Cambo, B., … Colla, S. (2022). Stem cell architecture drives myelodysplastic syndrome progression and predicts response to venetoclax-based therapy. Nature medicine, 28(3), 557–567. 

3. Kouros-Mehr, H., Slorach, E. M., Sternlicht, M. D., & Werb, Z. (2006). GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell, 127(5), 1041–1055. 

4. Bertucci, F., Houlgatte, R., Benziane, A., Granjeaud, S., Adélaïde, J., Tagett, R., Loriod, B., Jacquemier, J., Viens, P., Jordan, B., Birnbaum, D., & Nguyen, C. (2000). Gene expression profiling of primary breast carcinomas using arrays of candidate genes. Human molecular genetics, 9(20), 2981–2991. 

5. Tanaka, H., Takizawa, Y., Takaku, M., Kato, D., Kumagawa, Y., Grimm, S. A., Wade, P. A., & Kurumizaka, H. (2020). Interaction of the pioneer transcription factor GATA3 with nucleosomes. Nature communications, 11(1), 4136. 

6. Thway K. (2019). Well-differentiated liposarcoma and dedifferentiated liposarcoma: An updated review. Seminars in diagnostic pathology, 36(2), 112–121.