Emerging insights of tumor heterogeneity and drug resistance mechanisms in lung cancer targeted therapy

Abstract

The biggest hurdle to targeted cancer therapy is the inevitable emergence of drug resistance. Tumor cells employ different mechanisms to resist the targeting agent. Most commonly in EGFR-mutant non-small cell lung cancer, secondary resistance mutations on the target kinase domain emerge to diminish the binding affinity of first- and second-generation inhibitors. Other alternative resistance mechanisms include activating complementary bypass pathways and phenotypic transformation. Sequential monotherapies promise to temporarily address the problem of acquired drug resistance, but evidently are limited by the tumor cells' ability to adapt and evolve new resistance mechanisms to persist in the drug environment. Recent studies have nominated a model of drug resistance and tumor progression under targeted therapy as a result of a small subpopulation of cells being able to endure the drug (minimal residual disease cells) and eventually develop further mutations that allow them to regrow and become the dominant population in the therapy-resistant tumor. This subpopulation of cells appears to have developed through a subclonal event, resulting in driver mutations different from the driver mutation that is tumor-initiating in the most common ancestor. As such, an understanding of intratumoral heterogeneity-the driving force behind minimal residual disease-is vital for the identification of resistance drivers that results from branching evolution. Currently available methods allow for a more comprehensive and holistic analysis of tumor heterogeneity in that issues associated with spatial and temporal heterogeneity can now be properly addressed. This review provides some background regarding intratumoral heterogeneity and how it leads to incomplete molecular response to targeted therapies, and proposes the use of single-cell methods, sequential liquid biopsy, and multiregion sequencing to discover the link between intratumoral heterogeneity and early adaptive drug resistance. In summary, minimal residual disease as a result of intratumoral heterogeneity is the earliest form of acquired drug resistance. Emerging technologies such as liquid biopsy and single-cell methods allow for studying targetable drivers of minimal residual disease and contribute to preemptive combinatorial targeting of both drivers of the tumor and its minimal residual disease cells.

Keywords: Acquired drug resistance; Adaptive evolution; Minimal residual disease; Tumor heterogeneity.

Fig. 1

Models of drug resistance mechanisms following cancer targeted therapy. The EGFR-mutant model of drug resistance in lung cancer is shown here as an example. There are two recognized models of mechanisms of drug resistance known as preexisting mutations and adaptive evolution. In the preexisting mutations model, certain tumor cells growing within the parental population already have a survival advantage due to a preexisting mutation that can resist the targeting agent. Under continuous drug treatment, cells harboring the resistant mutation survive and proliferate to become the dominant clone, resulting in clinical drug resistance and tumor progression. Drug withdrawal at this point does not readily change the molecular makeup the cells. By contrast, in the adaptive evolution model, most tumor cells begin with a level playing field, with the exception of a subpopulation that may have been primed to activate prosurvival signaling pathways by an unknown regulatory or selective mechanism. While the majority of cells die under continuous drug treatment, a small subpopulation within the originally drug-sensitive cells will escape their initial dependence on the driver mutation, despite ostensibly identical genotype/genomic milieu, by adaptively altering either their transcriptome, signaling, or epigenome in a directed effort to survive against therapeutic pressure. This reprogramming process engenders the drug-escaping cells to enter into proliferative and metabolic quiescence. These adaptively resistant cells eventually acquire and accumulate mutations advantageous for further proliferative growth and the tumor progresses in fulminant resistance. In both aforementioned cases, the residual disease cells grow into a completely different tumor than the original under therapeutic pressure. However, previous work in vitro has demonstrated that early drug withdrawal can revert the adaptively resistant cells back to their parental, drug-sensitive state. This observation highlights the need for studying early adaptive resistant tumor cell populations and the mechanisms governing their shift to acquired resistance

Fig. 2

Landscapes of known molecular mechanisms of acquired targeted drug resistance to first- and second-generation EGFR-TKIs in lung cancer. The frequencies of each known mechanism are estimates acquired from studies based on tumor rebiopsies and repeat molecular tumor genotyping/genomic profiling at the time of acquired drug-resistant progression. The discovery of various mechanisms of acquired drug resistance further highlights the issues of tumor heterogeneity and adaptability of tumor cells to therapeutic pressure

Fig. 3

Spatial and temporal heterogeneity in tumor evolution. A single tumor tissue biopsy is equivalent to taking a mere “snapshot” of the molecular makeup of the tumor at a fixed time. The tumor’s evolutionary history and future as a result of progression and/or treatment would be missing from this single snapshot. Instead, serial and longitudinal tissue biopsies that track and follow the tumor’s development under therapy and during progression would empower more comprehensive and accurate representation of the tumor’s evolution, especially in exposing the conditions surrounding the emergence of subclones (as indicated by the different colors). Identification of subclones with known drug-resistant drivers can better inform the course of treatment most suitable for the tumor at its current state

Fig. 4

Conditions under which minimal residual tumor cells in molecular drug resistance can emerge. (1) Intrinsic resistance describes the cells’ inherent ability to resist the drug during initial therapy with preexisting stable genetic/genomic drug-resistant alteration(s). Shown are pretreatment lung adenocarcinoma cells harboring only the activating EGFR L858R mutation and cells that are double mutant for EGFR L858R and T790M. The T790M-mutants can survive initial treatments with an EGFR inhibitor (EGFRi) erlotinib or gefitinib, leading to incomplete response and eventual therapy failure and tumor progression stemmed from the expansion of the T790M clones. (2) Tumor cells adapt under therapeutic pressure to activate the early adaptive drug resistance program, engaging a cellular omics reprogramming scheme such as shift or modulation of prosurvival signaling, EMT-ness, cancer stemness and plasticity, glycolytic Warburg genes, among other undiscovered mechanisms. Drug-resistant molecular residual disease cells emerge as a result. As shown here in illustration, the STAT3/BCL-2/BCL-xL mitochondrial prosurvival signaling concurrent with hyperactivation of the TGFβ signaling pathway promote a drug-tolerant state that enables drug persistence during initial EGFR-TKI treatment. (3) The tumor microenvironment potentially contributes to the adaptive evolution of the tumor cells, resulting in minimal residual disease. As illustrated, stromal cells surrounding lung adenocarcinoma cells that secrete high levels of TGFβ have been known to stimulate the TGFβ axis in tumor cells via autocrine or paracrine signaling, granting them independence from EGFR signaling. TGFβ signals through IL-6, gp130, and JAK2 to stimulate STAT3 homodimerization. (4) Pharmacologic limitations, dose-limiting toxicities, or tumor intrinsic barriers can result in poor drug penetration into the tumor, resulting in pharmacokinetic therapy failure