NSD2 targeting reverses lineage plasticity and drug resistance in advanced prostate cancer [bulk RNA-seq]

Treatment with highly potent androgen receptor signaling inhibitors (ARSIs), such as enzalutamide and abiraterone promotes lineage plasticity in metastatic castration-resistant prostate cancer (mCRPC), which results in intra-tumor heterogeneity and emergence of mCRPC subtypes. The histological transformation from adenocarcinoma to aggressive neuroendocrine prostate cancer (CRPC-NE) has been associated with a loss of dependency on lineage-survival signals, leading to targeted drug resistance. Epigenomic reprogramming might be a fundamental driver of lineage plasticity. To determine CRPC-NE vulnerabilities, we have performed image-based screening using a small library of antibodies targeting different histone marks on CRPC-NE organoids derived from genetically engineered mice. We find that the histone mark H3K36me2 and the histone methyltransferase NSD2 (also known as MMSET/WHSC1) play important roles in maintaining the state of CRPC-NE. Knockout of NSD2 or ablation of H3K36me2 using H3.3K36M oncohistone reverted CRPC-NE to CRPC-AR-like phenotype. Simultaneous profiling of the transcriptome and epigenome from single cells collected from control (sgCtrl) and NSD2 knockout (sgNSD2) organoids, or from empty vector (EV) and H3.3K36M transduced organoids confirms the lineage reversal of treatment-resistant cells and maps the re-establishment of canonical AR signaling dependency. Moreover, H3K36me2 or NSD2 depleted mouse and human CRPC-NE organoids responded to enzalutamide treatment in vitro and in vivo, suggesting a restoration of ARSI sensitivity. Most importantly, a small molecule inhibitor of NSD2, which is similar to KTX-1001 (NCT05651932), in combination with enzalutamide leads to growth suppression or apoptosis of mouse and human CRPC-NE organoids, organoids of other CRPC subtypes and xenografts in vitro and in vivo. In conclusion, inhibition of NSD2 reverses lineage plasticity and reverts the state of treatment-resistant cells back into an ARSI-sensitive state. Thus, we suggest that the combination of NSD2 inhibition with AR inhibition may represent a novel therapeutic approach for patients with CRPC-NE and other CRPC subtypes. Overall design: The mouse NE tumors derived from NPp53 (Nkx3.1CreERT2/+; Ptenflox/flox; TrpP53flox/flox; Rosa26-EYFP) mice were cultured as organoids. To detect intercellular heterogeneity within organoids, I used single-cell RNA sequencing (scRNA-seq) to analyze lineage diversity of five mouse NE tumor organoids on passage 2. I also used single-nucleus ATAC sequencing (snATAC-seq) to profile the chromatin accessibilities of NPPO-1 organoids on passage 8, a multilineage NE tumor organoid line. The non-NE tumor cells from NPPO-1 organoids on passage 3 were isolated by fluorescence-activated cell sorting (FACS) according to a higher side scatter (SSC) value in non-NE compared to NE tumor cells. Some isolated non-NE tumor cells were analyzed by scRNA-seq to detect any contamination by NE tumor cells. Other isolated non-NE tumor cells were transduced with H2B-RFP lentivirus without antibiotic selection. H2B-RFP labeled tumor cells were either cultured alone or co-cultured with NE tumor cells to induce non-NE to NE conversion. To trace cell lineage of H2B-RFP-labeled cells, organoids from either monoculture or co-culture on passage 4 were analyzed by scRNA-seq (gene name: addgene26001). To quantitively compare the histone mark expression between NE and non-NE cells, epigenomic profiling using Cut&Tag was performed on 4 NE and 4 non-NE organoids derived from the same NPp53 background. To accurately quantify gene regulation, bulk RNA-seq was performed alongside epigenomic profiling on the organoids. To study the effects of NSD2 on NE lineage maintenance, we knockout of NSD2 on two mouse NE tumor organoids using CRISPR/Cas9. After antibiotic selection for 14 days, positive-transfected cells were isolated by FACS sorting according to the presence of RFP reporter signal. We used multiome single-nucleus ATAC and RNA sequencing (multiome snATAC + snRNA-seq) to analyze changes of cell lineage and state in sgNSD2 transfected NE tumor organoids in compared with sgControl (sgCtrl) organoids. To provide direct evidence of H3K36me2 histone modification in modulating NE lineage, mouse NE tumor organoids were transduced with lentivirus carrying the gene encoding the H3.3K36M oncohistone that can cause significant reduction in global levels of H3K36me2. After antibiotic selection for 14 days, cells positively transfected with H3.3K36M or empty vector (EV) control were analyzed using multiome snATAC + snRNA-seq to detect changes in cell lineage and state. In parallel, quantitative epigenomic profiling using Cut&Tag was performed on sgCtl and sgNSD2, or EV and H3.3K36M organoids to quantify global epigenomic changes. We have synthesized a small molecular inhibitor of NSD2 (NSD2i), which is similar to KTX-1001 (NCT05651932) in phase 1 clinical trial in patients with relapsed and refractory multiple myeloma. To evaluate if NSD2i recapitulates the effects of genetic interventions in reversal of lineage plasticity and restoring androgen receptor signaling inhibitor (ARSI) sensitivity, 1 µM NSD2i or DMSO was applied every other day on NPPO-1NE organoids cultured in a medium without DHT for 12 days. Organoids treated with either DMSO or 1 µM NSD2i were then collected for multiome snATAC + snRNA-seq analysis or Cut&Tag epigenomic profiling.