Extended Figure 10. Model of the role for elevated NSD3 H3K36 methylation activity in LUSC pathogenesis.

LUSC is characterized by a number of driver mutations such as increased expression of PI3K and SOX2, PI3K activating mutations, deletion of PTEN and other deletions and alterations. One of the more common genetic alterations is amplification of the 8p11-12 genomic region (~20% of LUSC patients). While increased expression of FGFR1 is postulated to be the causative mutation of the 8p11-12 amplicon, our work implicates amplification of the neighboring gene NSD3 as the main driving alteration. As shown, NSD3 amplification leads to increased NSD3 expression and hence increased synthesis of H3K36me2, which works in concert with other LUSC driver mutations to promote LUSC pathogenesis. In addition, as shown in Extended Data Fig. 2i, NSD3 overexpression is detected in 60% of LUSC patients, thus NSD3 is frequently highly expressed in LUSC that does not harbor the 8p11-12 amplifications. We also describe NSD3_T1232A as a gain-of-function (GOF) variant, that while far less common than the 8p11-12 amplicon and NSD3 overexpression, is an alteration present in human LUSC. The NSD3_T1232A variant, due to enhanced catalytic behavior, functionally acts like amplified or overexpressed NSD3 in increasing H3K36me2 synthesis and cooperating with other LUSC driver mutations to accelerate tumorigenesis. The increase in H3K36me2 by NSD3 overexpression or NSD3_T1232A reprograms the chromatin landscape, blocking synthesis of H3K27me3 and increasing H3K36me2, which stimulates transcription of key oncogenic targets including genes involved in mTOR signaling and MYC-associated pathways. We speculate that NSD3-regulated tumors become addicted to H3K36me2-driven transcriptional activation, rendering these tumors particularly vulnerable to BETi as NSD3 and BRD4 interact and cooperate in transcription. This hypersensitivity could potentially be exploited clinically due to an expanded therapeutic window for BETi and by using these drugs as targeted therapy for the tens of thousands of patients that are 8p11-12 positive. Not shown in the model, NSD3 is overexpressed in many LUSC samples without an underlying known alteration and we speculate that in such cases, increased NSD3 would contribute to tumorigenesis in a similar fashion as the scenarios described above in which NSD3 is hyperactive or overexpressed due to amplification.