Does Epigenetic Reprogramming Underlie Cancer Cell Lineage Plasticity?

As a hallmark of cancer development (Le Magnen et al. and Davies et al.), lineage plasticity supports the evolution of castration-resistant and often metastatic disease forms into neuroendocrine prostate cancer (Watson et al., Beltran et al., and Tang et al.). Unfortunately, this subtype of prostate cancer displays heightened aggressiveness and resistance to androgen receptor (AR) pathway inhibitors and suffers from poor clinical outcomes (Beltran et al., Zou et al., and Beltran et al.). A wide range of studies has suggested that epigenetic reprogramming mediates lineage plasticity in castration-resistant prostate cancer (CRPC) cells (Le Magnen et al., Beltran et al., and Quintanal-Villalonga et al.); therefore, treatment strategies against this hugely problematic disease form may look to integrate drugs that impact epigenetic regulatory layers, such as histone modifications. A team of researchers headed by Chao Lu and Michael M. Shen (Columbia University) sought to explore a role for epigenetics in cell plasticity and treatment resistance in neuroendocrine CRPC; now, their new Nature article reveals how the inhibition of the histone H3 lysine 36 dimethylation (H3K36me2) methyltransferase NSD2 (Bennett et al.) may form a component of novel therapeutic strategies (Li and Vasciaveo et al.).

Exploring links between the gene expression programs underpinned by histone modification profiles and cancer cell lineage plasticity at the single-cell level may provide insight into tumor evolution and the development of effective therapeutic strategies. Could Paired-Tag technology from Epigenome Technologies, which generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays, support these lofty research aims?

Therapeutic Implications of NSD2-H3K36me2 Methyltransferase-mediated Prostate Cancer Cell Lineage Plasticity

In the first part of this exciting new study, the authors created CRPC tumor organoids with neuroendocrine features, inherent heterogeneity, and insensitivity to AR pathway inhibitors (Zou et al.). Lineage-tracing experiments in organoids demonstrated the transdifferentiation of cells from an androgen receptor-expressing mesenchymal state to a neuroendocrine state, recapitulating observations of cell lineage plasticity in tumors in vivo (Zou et al.). Comparing histone modification levels in neuroendocrine and non-neuroendocrine cells from organoids revealed notable differences in H3K36me2 levels, while Western blotting provided evidence for the upregulation of the H3K36me2 methyltransferase NSD2 (Kuo et al. and Martinez-Garcia et al.) in neuroendocrine cells. Subsequent genome-wide profiling of histone modifications using CUT&Tag combined with bulk RNA-sequencing in neuroendocrine cells revealed increased levels of H3K36me2 at enhancer and promoter regions associated with neuroendocrine gene expression, consistent with the generally accepted link between this histone modification and transcriptional permissivity/accessible chromatin states.

.

In parallel, analyses of human prostate tumor samples (Beltran et al. and Zaidi et al.) associated NSD2 overexpression and upregulated H3K36me2 levels with neuroendocrine CRPC development; furthermore, an evaluation of human prostate cancer data revealed that high NSD2 expression correlated significantly with poor overall patient survival. Overall, the authors provided evidence supporting an association between NSD2 expression/high H3K36me2 levels at gene regulatory regions and neuroendocrine CRPC, as well as a potential link to cancer cell lineage plasticity.

The second half of this study focused on the consequences of the therapeutic modulation of NSD2 activity in CRPC cells. Initial analyses following CRISPR-mediated targeting of NSD2 in mouse CRPC organoids revealed the conversion of neuroendocrine cells to an AR-expressing adenocarcinoma phenotype and a substantial reduction in H3K36me2 levels, which rendered neuroendocrine cells more responsive to AR pathway inhibition. To explore the possibility that pharmacological NSD2 inhibition could reverse lineage plasticity and restore AR pathway sensitivity in prostate cancer patients, the authors evaluated a first-in-class NSD2 inhibitor, currently in an early-phase clinical trial for multiple myeloma treatment (KTX-1001; hereafter, NSD2i) (Deng et al.). This highly specific inhibitor substantially reduced H3K36me2 levels in mouse neuroendocrine organoid cells, leading to increased AR target gene expression and increased sensitivity to AR pathway inhibition, suggesting the reversal of cancer cell plasticity.

In agreement, an analysis of human neuroendocrine CRPC organoid cells revealed the substantial loss of H3K36me2 in response to NSD2i treatment and, excitingly, the anti-tumor synergism of NSD2i and AR pathway inhibitor cotreatment. NSD2i treatment of subcutaneous grafts of human CRPC organoids in mice prompted reduced H3K36me2 levels (with no adverse effects on mouse body weight) and modest effects on tumor growth; however, cotreatment with NSD2i and an AR pathway inhibitor resulted in robust growth suppression, with signs of a loss of neuroendocrine phenotypes, accompanied by overt necrosis and fibrosis and increased adenocarcinoma features.

NSD-mediated Histone Modifications and Lineage Plasticity: Moving Beyond Prostate Cancer?

This exciting study demonstrates the importance of NSD2 and H3K36me2 in regulating cell lineage plasticity and therapeutic resistance to AR pathway inhibition in neuroendocrine CRPC cells through epigenetic reprogramming of gene expression. In addition to providing a rationale for combining NSD2 and AR pathway inhibition in prostate cancer to reverse lineage plasticity and improve therapeutic responses, these data also highlight the potential of exploring NSD1 and NSD3 (NSD2-related genes/proteins) as drivers of cell plasticity in tumors such as head and neck cancer and squamous cell lung cancer (Papillon-Cavanagh et al. and Yuan et al.).

The implementation of Paired-Tag technology from Epigenome Technologies, which generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays, has the potential to provide deeper insight into studies such as these. What more could the simultaneous analysis of histone modifications and neuroendocrine gene expression in the same single cell tell us about prostate cancer cell lineage plasticity and therapeutic resistance? Stay tuned to the Epigenome Technologies blog to find out!