We use biochemistry and forward genetics to discover protein targets for small molecules with anti-cancer activity:

  • Using photochemistry and click chemistry to identify proteins that directly interact with the small molecule
  • Using a mammalian cancer cell line as a forward-genetic tool to discover compound-resistant alleles

We have identified numerous small molecule targets involved in different biological pathways including fatty acid metabolism, cholesterol biosynthesis, DNA replication, and pre-mRNA splicing.

      Hematological malignancies with high DCAF15 expression

      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 anti-cancer 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.

      Currently 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 anti-cancer 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) confer resistance to the toxic effects of indisulam in cultured cancer cells and in mice harboring tumor xenografts.   In the presence of indisulam, RBM39 associates with the CUL4-DDB1-DDA1-DCAF15 E3 ubiquitin ligase complex, leading to polyubiquitination and proteosomal degradation of RBM39.  Mutations in RBM39 that cause indisulam resistance, in contrast, do not associate with CUL4-DCAF15 and are thus neither modified with poly-ubiquitin nor degraded by the proteasome.

      In experiments with purified recombinant proteins, we found that indisulam forms 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 impede the formation of this complex.  Interestingly, we found that two other clinically tested sulfonamides with structural similarity to indisulam,  tasisulam and chloroquinoxaline s ulfonamide (CQS), share the same mechanism of action as indisulam.   RBM39 is a nuclear protein that is involved in 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 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 (for 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 anti-cancer drugs that act as a “molecular glue” bringing together the E3 ubiquitin ligase receptor Cereblon and a variety of neo-substrates.  In an analogous manner, SPLAM derivatives potentially could be used to target DCAF15 to novel neo-substrates that, like RBM39, are otherwise undruggable. 

      A major focus of our laboratory is to follow up these studies by:

      • Determine how new sulfonamides can be used to target proteins other than RBM39 in a DCAF15 dependent manner
      • Identify anticancer small molecules which act like “molecular glue” degraders
      • Identify how SPLAMs influence the degradation of endogenous DCAF15 substrates
      Cancer cell death

      CD437 is a retinoid-like small molecule that selectively induces apoptosis in cancer but not normal cells through an unknown mechanism. We used a forward genetic strategy to discover mutations in POLA1 that coincide with CD437 resistance (POLA1R). Introduction of one of these mutations into cancer cells by CRISPR/Cas9 genome editing conferred CD437 resistance demonstrating causality. POLA1 encodes DNA polymerase α, the enzyme responsible for initiating DNA synthesis during the S phase of the cell cycle. CD437 inhibits DNA replication in cells and recombinant POLA1 activity in vitro. Both effects are abrogated by mutations associated with POLA1R. In addition, we detected an increase in the total fluorescence intensity and anisotropy of CD437 in the presence of increasing concentrations of POLA1 consistent with a direct binding interaction. The discovery of POLA1 as the direct anti-cancer target for CD437 has the potential to catalyze its development into an anti-cancer therapeutic.

      There are therapeutic implications for CD437 as a newly discovered inhibitor of POLA1 and DNA replication. Many cancer drugs act by targeting any of a variety of aspects of DNA replication. Three classes of agents block DNA replication by influencing the template: anthracyclines through DNA intercalation, topoisomerase inhibitors through alteration of the supercoiled state of DNA, and alkylators through covalent modification of DNA. Other approved drugs inhibit replication by affecting nucleotides, which are essential for DNA synthesis. For instance, hydroxyurea, methotrexate, and pemetrexed reduce the overall level of nucleotides, and gemcitabine and cytarabine block replication by substituting for deoxycytidine. Finally, drugs that target enzymes that regulate DNA replication, such as inhibitors of cyclin-dependent kinases (CDK4 and CDK6), also have therapeutic efficacy. Surprisingly, despite the demonstrable clinical utility of anti-cancer therapies that target essential components of DNA replication, there are no currently available drugs that directly inhibit DNA polymerase.

      A major goal in our laboratory is to build on our discovery and develop and test DNA polymerase alpha inhibitors in pre-clinical models of cancer. The goal of the project is to develop first-in-class DNA polymerase alpha for the treatment of cancer.

      Targeted cancer therapies effects on tumor

      A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic to the same four of 12 human lung cancer cell lines at low nanomolar concentrations. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible stearoyl CoA desaturase (SCD) inhibitors. SCD is recognized as a promising biological target in cancer and metabolic disease. However, SCD is essential to sebocytes, and accordingly SCD inhibitors cause skin toxicity. Mouse sebocytes were unable to activate the benzothiazoles or oxalamides into SCD inhibitors, providing a therapeutic window for inhibiting SCD in vivo. We thus offer a strategy to target SCD in cancer by taking advantage of high CYP expression in a subset of tumors. These discoveries offer an opportunity to target SCD in either liver or tumors that express CYP4F11.

      Join Our Lab

      If you are interested in joining our research, please email Dr. Nijhawan.