Inducible drug modification: A new form of resistance

Resistance to chemotherapy is a major problem facing cancer treatment. Understanding the molecular basis for drug resistance has lagged behind our understanding and identification of new pathways to target. Even early on in treatment, patients can become resistant to therapy. For instance, substantial numbers of acute myeloid leukemia (AML) patients will not respond to the first round of standard therapy. While it is important to assess the different molecular events that drive cancer, and target these as part of personalized medicine, it is equally important to further understand why patients do not respond. Ultimately, we must understand these treatment failures in order to make future successes.

We took this approach to better understand why targeting the eukaryotic translation initiation factor eIF4E in AML patients with ribavirin worked dramatically well in many patients, but did not in others, and why all responding patients eventually relapsed. These studies led us to identify a novel form of drug resistance and excitingly, a means to overcome it.¹

Our study focused on the eukaryotic translation initiation factor eIF4E which is a potent oncogene that is elevated in approximately 30% of human cancers including M4 and M5 subtypes of AML.^1,2 eIF4E regulates the translation and nucleo-cytoplasmic export of a subset of transcripts that are essential for proliferation, survival and malignant transformation. Since association with the 7-methyl guanosine (m⁷G) cap structure at the 5′ end of mRNAs is essential for its functions,² targeting eIF4E with competitive inhibitors of the m⁷G, such as ribavirin,was undertaken.² Previous studies by us and others showed that ribavirin potently inhibited eIF4E activity in model systems.^2,3 Our Phase II ribavirin monotherapy clinical trial in poor prognosis AML patients showed that ribavirin led to a significant clinical improvement with a 45% objective response rate (remissions and blast responses).⁴ Although striking for this poor prognosis population, all responding patients eventually became resistant to ribavirin and some never responded despite having elevated eIF4E levels.⁵

In order to understand the molecular mechanisms underlying primary and acquired resistance, we generated cell line models and found 2 mechanims of resistance. First, we observed defective ribavirin uptake due to impaired drug influx and metabolism. Specifically, we identified down regulation of the key enzyme involved in ribavirin pro-drug metabolism and/or ribaivirn nucleoside transporter ENT1.⁴ Drug metabolism modulation is commonly observed in drug resistance, where drug uptake is abrogated, drug efflux is enhanced (e.g., MDR), or pro-drug metabolism is impaired. Although there have been attempts to target MDR transporters, this has not led to significant clinical benefit as of yet, mainly due to the substantial side effects observed with these therapies.

Since only 2 of our patients (over 15 studied), showed any defects in the transporter or pro-drug metabolism factors, we further explored mechanisms that could allow us to understand what were the factors driving drug resistance in our patients. Strikingly, we identified models where ribavirin uptake was normal, but its interaction with eIF4E was lost. Subsequent analysis indicated that the Glioma associated transcription factor Gli1 was highly elevated in these models and unexpectedly, this correlated with increased protein production of the UGT1A enzymes (Fig. 1). UGT1A enzymes are central to phase II drug metabolism where they add glucuronic acid (from the co-factor uridine disphoshate glucuronic acid, UDP-GA) to the drug leading to the loss of target-drug interactions and in some cases increased drug efflux.⁶ Here, we observed that ribavirin glucuronidation led to the loss of its interaction with eIF4E thereby underpinning subsequent resistance. Further, Gli1 elevation alone was sufficient to drive UGT1A protein production and drug resistance. Interestingly, Gli1-mediated glucuronidation also drove resistance to another unrelated drug, cytarabine, consistent with previous observations that UGT1As can modify many classes of drugs. Strikingly, addition of the FDA approved pharmacological inhibitors of Gli1 Visomdegib (GDC-0449)¹ or Gli1 knockdown in resistant cells led to a loss of glucuronidation, and drug resensitization. Thus, Gli1, via UGT1A, drives a novel form of multi-drug resistance which is targetable. In our patients, elevation of Gli1 and UGT1A levels were observed in patients who were primarily resistant and in patients at clinical relapse. Indeed, we observed that these factors were elevated in relapsed patients treated with standard AML therapies indicating that it could be a common form of drug resistance in these patients, and perhaps in other cancers as well. This form of drug resistance would be expected to affect a wide variety of drugs.

Generally, there are 3 means of drug resistance proposed: impaired drug net uptake (due to either increased efflux or decreased influx), mutation of the drug target or compensatory genetic re-wiring of the relevant pathways (Fig. 1).⁷ Here we present a fourth model, inducible drug modification. In this case, elevation of UGT1As would explain failure to treat refractory AML patients. We postulate that these modifications will not be limited to glucuronidation, with other aspects of phase II drug metabolism perhaps playing important roles in other forms of drug resistance. In clinical trials, many studies start with promising responses with patients quickly failing therapies. Perhaps this is not due to the fact that the wrong pathway was targeted or that the targeting was ineffectual, but rather to the rapid onset of drug resistance. Future studies focusing on drug resistance in parallel with the development of new therapies will likely lead to the development of the most efficacious therapies.

Figure 1. Well established molecular mechanisms underlying drug resistance include altered drug metabolism (increased drug efflux, decreased uptake), modification of drug targets, enhanced DNA repair, and genetic rewiring. In our novel mechanism, upregulation of Gli1 expression indirectly increases UGT1As, which catalyze the addition of glucuronic acid to the drug leading to the loss of drug-to-target interaction. This resistance could be reverted by either indirect or direct Gli1 inhibitors (GDC-0449 and Gant61, respectively). This mechanism might also apply to drugs other than Ribavirin and AraC.