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

Exploiting vulnerabilities in cancer signalling networks to combat targeted therapy resistance

Drug resistance remains one of the greatest challenges facing precision oncology today. Despite the vast array of resistance mechanisms that cancer cells employ to subvert the effects of targeted therapy, a deep understanding of cancer signalling networks has led to the development of novel strategies to tackle resistance both in the first-line and salvage therapy settings. In this review, we provide a brief overview of the major classes of resistance mechanisms to targeted therapy, including signalling reprogramming and tumour evolution; our discussion also focuses on the use of different forms of polytherapies (such as inhibitor combinations, multi-target kinase inhibitors and HSP90 inhibitors) as a means of combating resistance. The promise and challenges facing each of these polytherapies are elaborated with a perspective on how to effectively deploy such therapies in patients. We highlight efforts to harness computational approaches to predict effective polytherapies and the emerging view that exceptional responders may hold the key to better understanding drug resistance. This review underscores the importance of polytherapies as an effective means of targeting resistance signalling networks and achieving durable clinical responses in the era of personalised cancer medicine

Characterization and therapeutic exploitation of molecular vulnerabilities in genetically defined lung cancer

Lung cancer is one of the most common cancer types and responsible for the largest number of cancer-related deaths worldwide. Typically, lung cancer arises in individuals with heavy smoking background and only rarely in never-smokers. Various cells of origin within the lung give rise to distinct, molecularly heterogenous lung cancer subtypes with the two major subtypes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Targeted therapy options also vary significantly between the specific subtypes and while oncogene-driven lung adenocarcinoma (LUAD) is already successfully treated with targeted drugs, no targeted therapies are available in SCLC. LUAD is often driven genetic alterations such as point mutations and rearrangements in genes of receptor tyrosine kinases (RTKs) like EGFR leading to aberrant activation of receptor tyrosine kinase signaling and oncogenic transformation. Mutation-selective small molecule RTK inhibitors have been developed to specifically kill oncogene-addicted cancer cells. Introduction of third generation EGFR inhibitor osimertinib substantially increased survival of EGFR-mutant LUAD patients but on-target resistance mutations such as EGFR G724S limit osimertinib efficacy leading to tumor relapse. Remarkably, we observed that second-generation EGFR inhibitor afatinib displayed selective activity against EGFR G724S in cell line and animal models. In contrast to osimertinib, afatinib still binds to EGFR G724S and reduces cellular viability, EGFR signaling, transformation and in vivo growth of EGFR G724S cells, therefore providing a possible treatment strategy for patients that relapse after osimertinib treatment due to EGFR G724S. Oncogenic gene fusions involving RET also lead to cellular transformation and LUAD tumorigenesis. Previously, multi-kinase inhibitors were used to treat RET-rearranged cancers with limited success due to lack of RET-specificity and RET gatekeeper mutations impeding inhibitor binding. We identified AD80, a type II kinase inhibitor that binds RET in the DFG-out conformation. AD80 displayed selective activity against common RET fusions KIFB-RET and CCDC6-RET and retained activity against RET V804M gatekeeper mutation. AD80 efficiently reduced RET- and downstream signaling as well as RET-associated gene expression. AD80 also displayed in vivo efficacy in CCDC6-RET patient-derived xenograft (PDX) models, demonstrating the potential of type II inhibitors as targeted therapy against RET-rearranged LUAD. In contrast to NSCLC, SCLC is defined by inactivation of tumor suppressors TP53 and RB1 and lacks targetable oncogenic drivers. Frequent activation of MYC transcription factor family members (MYC, MYCL, and MYCN) further accelerate tumor growth and aggressiveness. We found that activation of individual MYC family members entails differential molecular vulnerabilities. MYC overexpression is associated with high levels of DNA damage, repression of BCL2 expression and high apoptotic priming, leading to higher sensitivity towards Aurora kinase and MCL1 inhibition whereas high MYCL/MYCN expression is associated with resistance against these perturbations. Our study highlights that MYC status can be predictive for therapy response and might be used for molecularly-guided, patient stratification for future targeted therapy regimens in SCLC. A rare but very aggressive lung cancer type, NUT carcinoma is driven by BRD4-NUT fusion protein leading to large-scale epigenetic reprogramming and deregulated transcription of genes driving tumorigenesis. Using high-throughput viability screening, we identified that NUT carcinoma cells are preferentially sensitive against CDK9 inhibition. We observed, that CDK9 inhibition increases RNA Polymerase II pausing possibly reverting BRD4-NUT-mediated, transcriptional activation of pro-tumor genes warranting further investigation of CDK9 inhibition in NUT carcinoma

Exploiting Synthetic Lethality and Network Biology to Overcome EGFR Inhibitor Resistance in Lung Cancer.

Despite the recent approval of third-generation therapies, overcoming resistance to epidermal growth factor receptor (EGFR) inhibitors remains a major challenge in non-small cell lung cancer. Conceptually, synthetic lethality holds the promise of identifying non-intuitive targets for tackling both acquired and intrinsic resistance in this setting. However, translating these laboratory findings into effective clinical strategies continues to be elusive. Here, we provide an overview of the synthetic lethal approaches that have been employed to study EGFR inhibitor resistance and review the oncogene and non-oncogene signalling mechanisms that have thus far been unveiled by synthetic lethality screens. We highlight the potential challenges associated with progressing these discoveries into the clinic including context dependency, signalling plasticity, and tumour heterogeneity, and we offer a perspective on emerging network biology and computational solutions to exploit these phenomena for cancer therapy and biomarker discovery. We conclude by presenting a number of tangible steps to bolster our understanding of fundamental synthetic lethality mechanisms and advance these findings beyond the confines of the laboratory

Combinatorial Drug Therapy for Cancer in the Post-Genomic Era.

Over the past decade, whole genome sequencing and other 'omics' technologies have defined pathogenic driver mutations to which tumor cells are addicted. Such addictions, synthetic lethalities and other tumor vulnerabilities have yielded novel targets for a new generation of cancer drugs to treat discrete, genetically defined patient subgroups. This personalized cancer medicine strategy could eventually replace the conventional one-size-fits-all cytotoxic chemotherapy approach. However, the extraordinary intratumor genetic heterogeneity in cancers revealed by deep sequencing explains why de novo and acquired resistance arise with molecularly targeted drugs and cytotoxic chemotherapy, limiting their utility. One solution to the enduring challenge of polygenic cancer drug resistance is rational combinatorial targeted therapy

Exploiting molecular vulnerabilities in genetically defined lung cancer models

Lung cancer is the leading cause of cancer-related death worldwide, with approximately 1.8 million deaths in 2020. Based on histology, lung cancer is divided into non-small cell lung cancer (NSCLC) (85 %) and small cell lung cancer (SCLC) (15 %). The most common types of NSCLC are lung squamous cell carcinoma (LUSC), large-cell carcinoma (LCC), and lung adenocarcinoma (LUAD). LUAD, the largest subgroup of NSCLC, is characterized by genomic alterations in oncogenic driver genes such as KRAS or EGFR. Mutations in the kinase domain of EGFR result in aberrant signaling activation and subsequent cancer development. Tyrosine kinase inhibitors (TKIs) selectively target and inhibit mutant kinases, thereby killing oncogene-addicted cancer cells. The introduction of TKIs into clinical practice shifted NSCLC treatment from cytotoxic chemotherapy towards precision medicine, improving both survival and the quality of life during therapy. Patients with canonical EGFR mutations like the point-mutation L858R or exon 19 deletions mutations, which account for the majority of EGFR mutations, respond well to EGFR targeted TKIs. However, rare mutations like insertions in exon 20 insertions still represent challenging drug targets. C-helix–4-loop insertion mutations in exon 20 push the C-helix into the active, inward position without altering the binding site for TKIs. This leaves the binding site for TKIs in kinases with exon 20ins mutations highly similar to wild type (WT) EGFR. Thus, the challenge in the development of exon 20 inhibitors is the design of wild type sparing small molecules. Here, we analyzed a novel small molecule EGFR inhibitor (LDC0496) targeting an emerging cleft in exon 20-mutated EGFR to achieve selectivity over the wild type. In contrast to classical EGFR TKIs, LDC0496 reduces the cellular viability of EGFR exon 20 mutated cells but spares wild type EGFR. Targeted therapy inevitably results in the development of on- or off-target resistance. Drug induced resistance mutations require the constant development of novel drugs targeting the diverse landscape of resistance mechanisms. We detected BRAF mutations in EGFR-driven lung cancer patients as a resistance mechanism to EGFR inhibitors. Notably, we also detected co-occurrence of EGFR and BRAF mutations before treatment start. Combination treatment of EGFR and mitogen-activated protein kinase kinase (MEK) inhibition displayed activity in BRAF- and EGFR-mutated xenograft studies, therefore providing a treatment strategy to overcome BRAF mutation as a resistance mechanism. Compared to NSCLC, SCLC lacks druggable targets and the initial chemosensitive state rapidly turns into a chemoresistance state. SCLC is genetically defined by a biallelic loss of tumor suppressors RB1 and TP53 and alterations of MYC family members. The transcription factor MYC is a challenging target that cannot be directly targeted. Therefore, alternative strategies are needed, for example targeting its co-factors, such as the MYC-interacting zinc finger protein 1 (MIZ1). To study the complex interplay of Myc–Miz1 in SCLC, we developed a novel mouse model with a truncated Miz1, which is unable to stably bind chromatin (RPMM: Rb1fl/flTrp53fl/flMycLSL/LSLMIZ1∆POZfl/fl). Compared to Miz1 wild type the characterization of the novel mouse model revealed tumor-onset, localization, size and immune infiltration to be unaffected by the ablation of the Miz1-POZ domain, but mice with Miz1-∆POZ live longer, exhibit an increased number of apoptotic cells and are more sensitive towards chemotherapy. We found that truncated Miz1 alter SCLC tumorigenesis towards a less aggressive phenotype and prolongs the chemosensitive state. Our study highlights alternative strategies to define novel vulnerabilities and options to overcome chemoresistance

Towards Precision Medicine in the Clinic: From Biomarker Discovery to Novel Therapeutics.

Precision medicine continues to be the benchmark to which we strive in cancer research. Seeking out actionable aberrations that can be selectively targeted by drug compounds promises to optimize treatment efficacy and minimize toxicity. Utilizing these different targeted agents in combination or in sequence may further delay resistance to treatments and prolong antitumor responses. Remarkable progress in the field of immunotherapy adds another layer of complexity to the management of cancer patients. Corresponding advances in companion biomarker development, novel methods of serial tumor assessments, and innovative trial designs act synergistically to further precision medicine. Ongoing hurdles such as clonal evolution, intra- and intertumor heterogeneity, and varied mechanisms of drug resistance continue to be challenges to overcome. Large-scale data-sharing and collaborative networks using next-generation sequencing (NGS) platforms promise to take us further into the cancer 'ome' than ever before, with the goal of achieving successful precision medicine

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.</p