Because of emergence of resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), no targeted treatments are available for patients with lung cancer who lose sensitivity due to new mutations or bypass mechanisms. We examined in animals and an alternative therapeutic approach making use of antibodies. An osimertinib-sensitive animal model of lung cancer, which rapidly develops drug resistance, has been employed. To overcome compensatory hyperactivation of ERK, which we previously reported, an anti-EGFR antibody (cetuximab) was combined with other antibodies, as well as with a subtherapeutic dose of osimertinib, and cancer cell apoptosis was assayed. Our animal studies identified a combination of three clinically approved drugs, cetuximab, trastuzumab (an anti-HER2 mAb), and osimertinib (low dose), as an effective and long-lasting treatment that is able to prevent onset of resistance to osimertinib. A continuous schedule of concurrent treatment was sufficient for effective tumor inhibition and for prevention of relapses. Studies employing cultured cells and analyses of tumor extracts indicated that the combination of two mAbs and a subtherapeutic TKI dose sorted EGFR and HER2 for degradation; cooperatively enhanced apoptosis; inhibited activation of ERK; and reduced abundance of several bypass proteins, namely MET, AXL, and HER3. Our assays and animal studies identified an effective combination of clinically approved drugs that might overcome resistance to irreversible TKIs in clinical settings. The results we present attribute the long-lasting effect of the drug combination to simultaneous blockade of several well-characterized mechanisms of drug resistance. .

Although two growth factor receptors, EGFR and HER2, are amongst the best targets for cancer treatment, no agents targeting HER3, their kinase-defective family member, have so far been approved. Because emergence of resistance of lung tumors to EGFR kinase inhibitors (EGFRi) associates with compensatory up-regulation of HER3 and several secreted forms, we anticipated that blocking HER3 would prevent resistance. As demonstrated herein, a neutralizing anti-HER3 antibody we generated can clear HER3 from the cell surface, as well as reduce HER3 cleavage by ADAM10, a surface metalloproteinase. When combined with a kinase inhibitor and an anti-EGFR antibody, the antibody completely blocked patient-derived xenograft models that acquired resistance to EGFRi. We found that the underlying mechanism involves posttranslational downregulation of HER3, suppression of MET and AXL upregulation, as well as concomitant inhibition of AKT signaling and upregulation of BIM, which mediates apoptosis. Thus, although HER3 is nearly devoid of kinase activity, it can still serve as an effective drug target in the context of acquired resistance. Because this study simulated in animals the situation of patients who develop resistance to EGFRi and remain with no obvious treatment options, the observations presented herein may warrant clinical testing.

Each group of the 56 receptor tyrosine kinases (RTK) binds with one or more soluble growth factors and coordinates a vast array of cellular functions. These outcomes are tightly regulated by inducible post-translational events, such as tyrosine phosphorylation, ubiquitination, ectodomain shedding, and regulated intramembrane proteolysis. Because of the delicate balance required for appropriate RTK function, cells may become pathogenic upon dysregulation of RTKs themselves or their post-translational covalent modifications. For example, reduced ectodomain shedding and decreased ubiquitination of the cytoplasmic region, both of which enhance growth factor signals, characterize malignant cells. Whereas receptor phosphorylation and ubiquitination are reversible, proteolytic cleavage events are irreversible, and either modification might alter the subcellular localization of RTKs. Herein, we focus on ectodomain shedding by metalloproteinases (including ADAM family proteases), cleavage within the membrane or cytoplasmic regions of RTKs (by gamma-secretases and caspases, respectively), and complete receptor proteolysis in lysosomes and proteasomes. Roles of irreversible modifications in RTK signaling, pathogenesis, and pharmacology are highlighted.

Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although the cure rate for this disease is greater than 85%, ALL remains the number one cause of cancer-related deaths in children due to relapse of ALL. Therefore, there is a great need to identify new therapies for patients who have recurrent disease. Recently, a subset of pediatric ALL patients whose leukemic cells express high levels of thymic stromal lymphopoietin receptor (TSLPR/CRLF2) have been shown to have an increased risk of relapse and shorter disease free and poorer overall survival. Overexpression of TLSPR occurs in 8% of unscreened pediatric precursor B ALL and occurs by genomic rearrangement of the TSLPR gene, which fuses the unmutated TSLPR gene to altered transcriptional control or by other yet to be described means. The mechanism by which Thymic Stromal Lymphopoietin (TLSP) signaling contributes to increased risk of relapse is unknown. Studies have shown aberrant signaling in high TSLPR expressing ALL patient derived cell lines relying heavily on in vitro experiments. As no pre-clinical model of high TSLPR ALL has been published, we created a model of high TSLPR expressing leukemia to study TSLPR overexpressing leukemia progression. We have created a high TSLPR expressing leukemia cell line through retroviral transduction of a transplantable syngeneic mouse leukemia model in which the leukemic progression can be studied with physiologic levels of TSLP. This high TSLPR leukemia has levels of expression of TSLPR comparable to what is found on human leukemia that overexpress TSLPR. The TSLPR is functional in these cells and we see increased phosphorylation of STAT5 protein in response to IL-7 or TSLP stimulation. When we introduce the leukemia into mice and look at disease progression, we observed an 8 fold difference in the numbers of cells in the bone marrow 5 days after intravenous injection corresponding to an early stage of leukemia progression (Figure 1. high TSLPR 1.61%+/-0.95 vs. low TSLPR 0.20%+/-0.16). Interestingly we find no significant difference in long term survival of mice injected with either low or high TSLPR leukemia lines. The increased numbers of leukemic cells in the bone marrow at early stages of leukemic progression could be due to an increased rate of proliferation or better survival/engraftment. Low and high TSLPR expressing cells show no significant difference in growth rate in vitro or in vivo in dye dilution assays (Figure 2) suggesting that the increase in leukemic cells in the bone marrow is through enhanced survival. To test this, we treated low and high TSLPR leukemia lines with the steroid dexamethasone in the absence or presence of TSLP. We found that the addition of TSLP significantly reduced the Annexin V positive relative to cells not treated with TSLP in the high TSLP expressing leukemia cells, while in low TSLPR expressing cells we observed no decrease in Annexin V positive cells (Figure 3). This suggests that high TSLPR expression sensitizes leukemia cells to TSLP in the leukemia microenvironment. To confirm that this is the case we have found by gene expression analysis that we can detect TSLP in mouse bone marrow. We hypothesize that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse. To test this hypothesis we have generated TSLPR-Fc conjugates to block TSLP signaling. We plan on using these reagents to block TSLP signaling to see if we can reverse the increased amounts of leukemia we find in mice at early stages of leukemic progression as well as the eliminate the survival advantage provided by TSLP to high TSLPR expressing leukemic cells in response to chemotherapeutic agents. Disclosures: No relevant conflicts of interest to declare.