Gene Editing and Multi-Omics Reveal Novel Metabolic Vulnerabilities in t(4;14)-Associated Multiple Myeloma

Background: Chromosomal translocation t(4;14) is a recurrent genetic lesion in multiple myeloma (MM) which drives aberrant overexpression of the histone methyltransferase NSD2 and is associated with a more guarded prognosis. Despite its oncogenic outcomes, NSD2 overexpression in MM cells could simultaneously confer cellular vulnerabilities that can be therapeutically exploited.

Aim: To define potential therapeutic susceptibilities driven by NSD2 overexpression in t(4;14) MM.

Methods: We performed genome-wide knockout screening in isogenic NSD2 high and low KMS11 cells using the Brunello CRISPR library, followed by validation of key target genes in several different t(4;14) MM cell lines.

Results: These screens revealed increased reliance of NSD2-overexpressing cells on multiple cellular processes including mitochondrial respiration, transcriptional elongation and proteasomal degradation. One of the top preferential dependencies in NSD2-high MM cells is the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). AK2 catalyzes the reversible interconversion of ADP to ATP and AMP. Cancer Dependency Map (depmap) analysis indicated that AK2 loss is detrimental to most MM cell lines but it is not a dependency in most solid tumor cells. Reliance on AK2 was modest in t(4;14) MM cells engineered to express low levels of NSD2 but the detrimental effect of AK2 depletion by shRNA or CRISPR was exacerbated upon enforced overexpression of NSD2. Gene expression studies showed that AK2 suppression leads to induction of genes involved in oxidative stress response. Metabolomic profiling of NSD2 high and low isogenic MM cells demonstrated striking reduction of NAD+ and elevation of nicotinamide (NAM) levels in NSD2 overexpressing cells, which correlates with a 10-fold induction of expression of the NAD+ consuming enzyme CD38 by NSD2. NAD+ and its metabolite NADP+ are essential co-factors of multiple antioxidant enzymes and its depletion can exacerbate oxidative damage. Therefore, NAD+ depletion likely accounts for the increased susceptibility of NSD2 high MM cells to AK2 loss.

In vitro cell proliferation/viability assays demonstrated increased sensitivity of NSD2 high MM cells to PI exposure suggesting that NSD2 overexpression drives a higher load of unfolded proteins, correlating with loss of NAD+ and redox system activity. AK2 suppression in MM cells also impairs activation of the adaptive unfolded protein response through the IRE1a-XBP1 axis leading to decreased protein folding capacity. The combination of high unfolded protein load and poor ATP shuttling to the endoplasmic reticulum (ER) secondary to AK2 loss may represent an additional mechanism of selective lethality. Furthermore, AK2 deficient MM cells displayed increased sensitivity to PIs.

Transcriptomic analyses in NSD2 high and low MM cells revealed that NSD2 overexpression, besides driving an oncogenic transcriptional program, alters the expression of several genes involved in nucleotide metabolism. Perturbed nucleotide metabolism was further documented by untargeted metabolite profiling which showed a notable depletion of several pyrimidine metabolites when NSD2 is overexpressed. Intriguingly, induced reduction of pyrimidine synthesis by treatment with DHODH inhibitors appeared to be more detrimental in NSD2 overexpressing MM cells.

Conclusions: NSD2 overexpression in MM cells alters cellular metabolism and confers higher susceptibility to oxidative stress and unfolded protein loads. By inducing the expression of NAD+ consuming enzymes, NSD2 overexpression results in NAD+ depletion which likely underlies the described vulnerabilities. Loss of the mitochondrial adenine nucleotide regulator AK2 is more detrimental to NSD2 overexpressing MM cells as it diminishes efficient ATP utilization in the ER perturbing the ability of MM cells to handle high loads of unfolded immunoglobulins and mitigate oxidative stress. Furthermore, integrated analyses of the different omics datasets highlighted other NSD2-driven liabilities, including mitochondrial respiration, transcriptional elongation and nucleotide metabolism, that can be targeted to improve prognosis of MM.

Mitsiades:H3 Biomedicine: Research Funding; BMS: Research Funding; Nurix: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Arch Oncology: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; Secure Bio: Consultancy; FIMECS: Consultancy; Ionis Pharmaceuticals: Consultancy; Fate Therapeutics: Consultancy; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; Novartis: Research Funding; Springworks: Research Funding; Oncopeptides: Consultancy.