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Towards evidence-based combinations of approved and novel cancer drugs

Periodic Reporting for period 2 - onCOMBINE (Towards evidence-based combinations of approved and novel cancer drugs)

Reporting period: 2019-04-01 to 2020-09-30

Lung cancer is widely recognised as the world leading cause of cancer-related death. The study we report herein addresses a major open issue related to the way patients with lung cancer are currently treated. The majority of patients present the non-small cell type of lung cancer (NSCLC). Patients with NSCLC are often grouped according to the type of mutations their tumours harbor. The major subtype expresses oncogenic forms of the RAS protein, but this group can be treated only with immune checkpoint blockers. The second largest subtype harbors diverse mutations within the gene encoding for EGFR. Studies reported in the last decade identified patients expressing EGFR mutants as a distinct group, highly responsive to tyrosine kinase inhibitors (TKIs). Unfortunately, despite initial dramatic activity of EGFR-specific TKIs, within 10-14 months almost all patients acquire resistance to the first-generation TKIs. Although newer generation drugs have been developed, resistance to the new drugs is almost inevitable. As a result, patients with mutant EGFR lung cancer who sequentially underwent treatment with the 1st-, 2nd- and 3rd-generation inhibitors have no viable treatment options other than chemotherapy. Hence, finding a suitable treatment for patients who evolved resistance to EGFR-specific TKIs is considered a most urgent medical challenge.
As an alternative to the sequential TKI treatment scenario, our studies of the last 3 years offer antibody-based treatment strategies. The following therapeutic scenarios were tested in animal models:
(i) Prior to the approval of ONCOMBINE, we applied a triple mixture of monoclonal antibodies, 3XmAbs, targeting EGFR, HER2 and HER3. Following the approval of this ERC project, we compared 3XmAbs and a third-generation TKI, osimertinib, and showed that simultaneous blockade of EGFR, along with its two siblings, HER2 and HER3, is both necessary and sufficient for preventing emergence of resistance to the newly approved TKI. Our biochemical and animal studies predicted synergy between osimertinib and 3XmAbs, which we verified in animals: a weakly active low dose of osimertinib enabled 3XmAbs to durably and completely eradicate already established drug-resistant tumours. Taken together, the data indicate that targeting the extracellular domain of EGFR and its sibling, by means of an antibody mixture, can overcome emergence of resistance to drugs targeting the intracellular kinase domain of EGFR.
(ii) Additional animal studies we performed identified a combination of just two antibodies, both are clinically approved drugs, cetuximab (an anti-EGFR antibody) and trastuzumab (an anti-HER2 mAb), in combination with the 3rd-generation TKI, osimertinib (low dose), as an effective and long-lasting treatment, able to prevent onset of resistance to osimertinib. However, continuous schedules of concurrent treatments were necessary for effective tumor inhibition and for prevention of relapses. Studies employing cultured cells and analyses of tumour extracts indicated that the combination of two mAbs and sub-optimal TKI concentrations sorted EGFR and HER2 for degradation, cooperatively enhanced apoptosis, inhibited activation of both ERK and AKT, and reduced abundance of several bypass proteins, namely as MET, AXL and HER3.
(iii) In the next step, we developed an upfront treatment, which might replace the sequential strategy. To this end, we simulated in animal models (both patient-derived xenografts and regular xenografts) the situation of TKI-naïve patients, who are diagnosed with NSCLC harbouring a single site EGFR mutation. Employing multiple in vivo and in vitro systems permitted head-to-head comparative studies that examined the efficacies of treatments comprising antibodies and a TKI previously approved as a 1st-line drug (i.e. erlotinib, afatinib or osimertinib) and a mixture of two clinically approved monoclonal antibodies specific to EGFR and its kin, HER2 (i.e. cetuximab and trastuzumab, respectively). Remarkably, the results indicated amazing superiority of all three TKI+antibody combos over the respective monotherapies, in terms of preventing acquired resistance. Prolonged treatments, high dose and narrow specificity of the TKI to EGFR were essential for the TKI-antibody synergy. Analyses of tumor specimens attributed drug cooperation to downregulation of receptors previously implicated in resistance to TKIs, elimination of survivin, as well as silencing of both ERK and AKT. Hence, we propose that upfront treatments with combinations of kinase inhibitors and antibodies (all are clinically approved) might replace the currently used sequential treatments with next-generation TKIs.
(iv) ONCOMBINE attempts yet another strategy, namely: enhancing responses of patients with EGFR mutations to immunotherapy. It is well documented that EGFR mutant tumours exhibit relatively low response rates to immune checkpoint blockers (ICBs). This is in marked contrast to the high response rates exhibited by KRAS mutant tumours. While response rates of patients correlate with high tumour mutational burden (TMB), this indicator is less predictive when applied to the EGFR mutant group of patients. Therefore, understanding mechanisms underlying resistance of EGFR mutant patients to immunotherapy is urgently needed. An important clue as to the mechanism driving resistance of the EGFR mutant group to ICBs has been provided by clinical observations made with patients expressing rare mutant forms of EGFR, who respond rather well to ICBs. Because treatment outcomes vary by EGFR allele, we assumed that features intrinsic to the tumour cells drive the relatively high resistance of the EGFR+ group. While studying intrinsic factors that potentially underlie immunosuppression, we discovered previously unknown strong physical and functional interactions between phospholipase C gamma (PLC-g) and PD-L1.
Currently, the field of lung cancer treatment is dominated by TKIs and ICBs. However, these therapeutic strategies benefit only relatively small fractions of patients and those who initially respond to treatment often develop resistance. ONCOMBINE aims at drug combinations that make use of TKIs, anti-receptor antibodies and ICBs. The results we collected so far highlight important features of such combinations, such as the ability of TKIs+mAbs combos to induce growth arrest states resembling senescence and eliminating resistance-conferring receptors, such as MET and AXL. We predict that the final outcome of the project will be applications of effective combinations of drugs able to delay onset of resistance on the basis of in-depth mechanistic understanding.