Indisulam is an aryl sulfonamide drug that inhibits the proliferation of certain human cancer cell lines. Its mechanism of action and the mechanism underlying its selectivity are poorly understood. On the basis of its anticancer activity in vitro and in mice, indisulam has been extensively tested in patients with advanced-stage solid tumors. No unacceptable toxicities were reported in patients receiving indisulam monotherapy, but fewer than 10% of patients showed a clinical response.

At present, there is no way to predict which cancer patients are most likely to benefit from indisulam treatment. We reasoned that a better understanding of the molecular mechanism underlying indisulam’s anticancer activity might reveal why only a subset of tumors respond to it. This in turn might lead to more effective clinical use of the drug. To study indisulam’s mechanism of action, we identified genetic mutations that confer resistance to its cytotoxic effect.

Using a forward genetic strategy, we discovered that several single amino acid substitutions in a nuclear protein called RBM39 (RNA binding motif protein 39) conferred resistance to the toxic effects of indisulam in cultured cancer cells and in mice with tumor xenografts. In the presence of indisulam, RBM39 associated with the CUL4-DDB1-DDA1-DCAF15 E3 ubiquitin ligase complex (CUL4-DCAF15), leading to polyubiquitination and proteasomal degradation of RBM39. Mutations in RBM39 that cause indisulam resistance, in contrast, did not associate with CUL4-DCAF15 and were thus neither modified with polyubiquitin nor degraded by the proteasome.

In experiments with purified recombinant proteins, we found that indisulam formed a ternary complex with RBM39 and the E3 ubiquitin ligase receptor DCAF15, with no detectable affinity for either species alone. RBM39 mutations that cause indisulam resistance impeded the formation of this complex. Interestingly, we found that two other clinically tested sulfonamides with structural similarity to indisulam—tasisulam and chloroquinoxaline sulfonamide (CQS)—share the same mechanism of action as indisulam. RBM39 is a nuclear protein that is involved in precursor mRNA (pre-mRNA) splicing. Biochemical isolation of RBM39 revealed an association with numerous splicing factors and RNA binding proteins. We found that degradation of RBM39 by indisulam led to aberrant pre-mRNA splicing, including intron retention and exon skipping, in hundreds of genes.

In a large survey of indisulam sensitivity across more than 800 cancer cell lines, we found that cancer cells derived from the hematopoietic and lymphoid (HL) lineages were more sensitive than cancer cells derived from other lineages. In HL cancer cell lines, DCAF15 mRNA expression levels and DCAF15 gene copy number variation directly correlated with indisulam sensitivity.

Cancer genome–sequencing studies have highlighted the importance of pre-mRNA splicing in tumorigenesis. Drugs such as indisulam, tasisulam, and CQS—which we collectively refer to as SPLAMs (splicing inhibitor sulfonamides)—provide a strategy to target RBM39-dependent pre-mRNA splicing in cancer. Many of the earlier clinical trials of indisulam focused on patients with solid tumors. Our findings suggest that indisulam may be most effective in patients with leukemias and lymphomas that express relatively high levels of DCAF15.

The activity of SPLAMs resembles that of IMiDs (immunomodulatory drugs). IMiDs are anticancer drugs that act as a “molecular glue,” bringing together the E3 ubiquitin ligase receptor cereblon and a variety of neosubstrates. In an analogous manner …