To Tyrosine Kinase Inhibitors Sensitivity Research Articles

Abstract 5843: Exploring the implications of LPAR-1 expression in TKI-resistant NSCLC

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer in the United States with a 5-year survival rate hovering between 20-30%. Mutations in EGFR can initially cause the tumors to respond to tyrosine kinase inhibitors (TKI). Osimertinib, a third generation TKI, is the preferred first or second line of treatment and despite being effective, recent reports indicate that patients develop resistance within 10-18 months of treatment. TKI resistance limits therapeutic options for patients and therefore, alternative strategies are being explored. In this context, Lysophosphatidic acid receptors (LPARs), a family of G-protein coupled receptors consisting of LPARs 1-6 expressed differentially in prostate, breast, ovarian, pancreatic, and brain cancers are being explored as potential targets for drug resistant cancers. Specifically, LPAR-1 has been shown to play a role in tumor cell clonogenicity, migration, and response to therapy in EGFR mutant NSCLC cells. Pharmacological inhibition or CRIPSR based deletion of LPAR-1 in EGFR mutant NSCLC cell lines HCC827 and NCI-H3255, increased TKI sensitivity by reducing clonogenic potential. Further, detailed pathway analysis in TKI sensitive and resistant EGFR mutant cell lines suggests that LPAR-1 expression influences drug sensitivity. Surprisingly, the role of LPAR-1 in TKI sensitivity is not well understood. In the present study, we have performed detailed investigation on the implications of LPAR-1 expression in the development of Osimertinib resistance. Methods: Osimertinib resistant EGFR mutant cell lines (OR) were generated using dose-escalation method and drug-tolerant persisters (DTPs) were generated using a short-term high exposure method. Gene and protein expressions were examined using qPCR and western blotting. Subcutaneous tumor cell derived xenografts were generated for in vivo studies. Results: LPAR-1 is downregulated in Osimertinib resistant NSCLC cells. Further, we also observe that LPAR-1 levels are lower in DTP cells, confirming a progressive loss of LPAR-1 with Osimertinib treatment. In vivo studies in cell derived xenografts correlated with the in vitro studies. Conclusion: Our studies indicate LPAR-1 downregulation correlates to Osimertinib resistance in EGFR mutant NSCLC cell lines. LPAR-1 downregulation has a key role in development of Osimertinib resistance. Future studies will focus on delineating LPAR-1 signaling pathways related to TKI resistance and investigating the effect of LPAR-1 overexpression using CRISPR technology. Citation Format: Varsha Srinivasan, Zhaohui Li, Raghuraman Kannan, Anandhi Upendran. Exploring the implications of LPAR-1 expression in TKI-resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5843.

Tyrosine kinase inhibitor response of ABL-class acute lymphoblastic leukemia: the role of kinase type and SH3 domain

AbstractAcute lymphoblastic leukemia (ALL) with fusions of ABL-class tyrosine kinase genes other than BCR::ABL1 occurs in ∼3% of children with ALL. The tyrosine kinase genes involved in this BCR::ABL1-like (Ph-like) subtype include ABL1, PDGFRB, ABL2, and CSF1R, each of which has up to 10 described partner genes. ABL-class ALL resembles BCR::ABL1-positive ALL with a similar gene expression profile, poor response to chemotherapy, and sensitivity to tyrosine kinase inhibitors (TKIs). There is a lack of comprehensive data regarding TKI sensitivity in the heterogeneous group of ABL-class ALL. We observed variability in TKI sensitivity within and among each ABL-class tyrosine kinase gene subgroup. We showed that ALL samples with fusions for any of the 4 tyrosine kinase genes were relatively sensitive to imatinib. In contrast, the PDGFRB-fused ALL samples were less sensitive to dasatinib and bosutinib. Variation in ex vivo TKI response within the subset of samples with the same ABL-class tyrosine kinase gene was not associated with the ALL immunophenotype, 5′ fusion partner, presence or absence of Src-homology-2/3 domains, or deletions of IKZF1, PAX5, or CDKN2A/B. In conclusion, the tyrosine kinase gene involved in ABL-class ALL is the main determinant of TKI sensitivity and relevant for specific TKI selection.

A single-cell atlas identifies pretreatment features of primary imatinib resistance in chronic myeloid leukemia

AbstractPrimary resistance to tyrosine kinase inhibitors (TKIs) is a significant barrier to optimal outcomes in chronic myeloid leukemia (CML), but factors contributing to response heterogeneity remain unclear. Using single-cell RNA (scRNA) sequencing, we identified 8 statistically significant features in pretreatment bone marrow, which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis [BC] transformation). Employing machine-learning, we identified leukemic stem cell (LSC) and natural killer (NK) cell gene expression profiles predicting imatinib response with >80% accuracy, including no false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor [ERP] expansion in vivo (P < .05), and a 2- to 10-fold (6.3-fold in group A vs 1.09-fold in group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared with myeloid progenitors (P < .001). These LSC features were lost with progressive resistance, and MYC- and IRF1-driven inflammatory regulons were evident in patients who progressed to transformation. Patients with MMR also exhibited a 56-fold expansion (P < .01) of a normally rare subset of hyperfunctional adaptive-like NK cells, which diminished with progressive resistance, whereas patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance. Finally, we developed antibody panels to validate our scRNA-seq findings. These panels may be useful for prospective studies of primary resistance, and in assessing the contribution of predetermined vs acquired factors in TKI response heterogeneity.

Comprehensive analysis of copy number variance and sensitivity to common targeted therapy in clear cell renal cell carcinoma: In silico analysis with in vitro validation.

BackgroundChromosomal rearrangements are common in clear cell renal cell carcinoma (ccRCC) and their roles in mediating sensitivity to tyrosine kinase inhibitors (TKIs) and mTOR inhibitors (mTORi) remain elusive.MethodsWe developed an in silico strategy by screening copy number variance (CNV) that was potentially related to TKI or mTORi sensitivity in ccRCC by reproducing the TCGA and GDSC datasets. Candidate genes should be both significantly prognostic and related to drug sensitivity or resistance, and were then validated in vitro.ResultsADCYAP1 loss and GNAS gain were associated with sensitivity and resistance and to Cabozantinib, respectively. ACRBP gain and CTBP1 loss were associated with sensitivity and resistance and to Pazopanib, respectively. CDKN2A loss and SULT1A3 gain were associated with sensitivity and resistance and to Temsirolimus, respectively. CCNE1 gain was associated with resistance to Axitinib and LRP10 loss was associated with resistance to Sunitinib. Mutivariate analysis showed ADCYAP1, GNAS, and CCNE1 remained independently prognostic when adjusted for the rest.ConclusionHere we show CNVs of several genes that are associated with sensitivity and resistance to commonly used TKIs and mTORi in ccRCC. Further validation and functional analyses are therefore needed.

Abstract IA10: Modeling sensitivity and resistance to systemic therapies in lung cancer

Abstract Targeted therapies have transformed the landscape for the treatment of metastatic lung cancer. Despite their success, targeted therapies are not curative and acquired resistance is a major impediment to cures for patients treated with these therapies. A paradigm for the success of targeted therapies in lung cancer comes from epidermal growth factor receptor (EGFR) mutant lung cancer where mutations in exons encoding the tyrosine kinase domain of EGFR confer sensitivity to tyrosine kinase inhibitors (TKIs), and several TKIs are currently approved for the first-line treatment of EGFR mutant lung cancer. Moreover, there is heterogeneity in the durability and depth of responses between patients and at different metastatic sites within individual patients. Therefore, there is a need to understand the biology of EGFR mutant tumors and identify factors that affect TKI sensitivity. We developed transgenic mouse models of EGFR mutant lung cancer and have used them to study tumor progression and TKI resistance. When we modeled acquired resistance to the TKI osimertinib as a first-line therapy in transgenic mouse models of EGFRL858R-induced lung adenocarcinoma, we found that it is mediated largely through secondary mutations in EGFR—either C797S or L718V/Q or Kras mutations. We also identified a therapeutic approach to prevent the emergence of secondary mutations in EGFR. To determine whether the presence of co-occurring genetic alterations contributes to a more complex spectrum of resistance mechanisms in vivo, we have developed new mouse models to specifically evaluate the consequences of co-occurring tumor suppressor gene alterations on the progression and TKI sensitivity of EGFR mutant tumors. Collectively, our findings highlight how genetically engineered mouse models of lung cancer, including those with complex genotypes, can be leveraged to study tumor progression and drug resistance in vivo. Citation Format: Katerina A. Politi. Modeling sensitivity and resistance to systemic therapies in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA10.

Inhibition of Skp2 Sensitizes Chronic Myeloid Leukemia Cells to Imatinib.

IntroductionSkp2 is an E3 ubiquitin ligase that plays an important role in modulating tumor progression. The mechanisms underlying Skp2 in the promotion of proliferation and its function in the primary resistance to tyrosine kinase inhibitors (TKIs) in human CML remain to be determined. This study aimed to investigate the function of Skp2 in CML progression as well as its effects on TKI sensitivity.MethodsExpression of Skp2 in leukocytes from patients with CML and normal blood samples was analyzed by qRT-PCR. Cell proliferation was analyzed by EdU incorporation and cell counting assays. Luciferase reporter and chromatin immunoprecipitation assays were used for examination of the effects of CREB on Skp2 expression. The apoptosis in vitro of K562 cells was analyzed by MTT and caspase 3/7 activity assays.ResultsThe present study demonstrates that Skp2 was expressed at a higher level in patients with CML compared with healthy donors, and the elevated expression of Skp2 is critical for CML cell proliferation. Mechanistically, Skp2 was transcriptionally upregulated by CREB responsive to the PI3K/Akt signaling pathway. Furthermore, inhibition of Skp2 expression by shRNAs or blocking the PI3K/Akt/CREB pathway greatly enhances the sensitivity of CML cells to Imatinib treatment.ConclusionWe conclude that the PI3K/Akt/CREB axis regulates the sensitivity of K562 cells to Imatinib via mediating Skp2 expression. The present study revealed an unknown role of Skp2 in CML progression and provided new aspects on the Skp2-modulated TKI sensitivity in CML, contributing to the development of potential therapeutic anticancer drugs.

Dioscin overcome TKI resistance in EGFR-mutated lung adenocarcinoma cells via down-regulation of tyrosine phosphatase SHP2 expression.

Resistance to tyrosine kinase inhibitors (TKIs) results in tumor relapse and poor prognosis in patients with lung adenocarcinoma. TKI resistance caused by epidermal growth factor receptor (EGFR) mutations at T790M and c-Met amplification occurs through persistent activation of the MEK/ERK and PI3K/AKT signaling pathways. We therefore expected that dual inhibitors of both signaling pathways could overcome TKI resistance in lung adenocarcinoma. Here, dioscin was selected from a product library of Chinese naturally occurring compounds and overcame TKI resistance in EGFR-mutated lung adenocarcinoma cells. Mechanistically, dioscin may down-regulate the expression of SH2 domain-containing phosphatase-2 (SHP2) at the transcription level by increasing p53 binding to the SHP2 promoter due to reactive oxygen species (ROS). Simultaneous inhibition of MEK/ERK and PI3K/AKT activation via decreased SHP2 expression and its interaction with GAB1 may be responsible for dioscin-mediated TKI sensitivity. A higher unfavorable response to TKI therapy occurred more commonly in patients with high SHP2 mRNA expression than in patients with low SHP2 mRNA expression. Therefore, we suggest that dioscin may act as a dual inhibitor of the MEK/ERK and PI3K/AKT signaling pathways to overcome TKI resistance via dysregulation of SHP2 expression in lung adenocarcinoma.

Establishment and characterization of patient-derived xenograft models of gastrointestinal stromal tumor resistant to standard tyrosine kinase inhibitors.

Gastrointestinal stromal tumors (GISTs) with KIT or platelet-derived growth factor receptor alpha (PDGFRa) oncogenic driver gene mutations, respond to tyrosine kinase inhibitors (TKIs) including imatinib, sunitinib, and regorafenib. However, most patients develop TKI resistance; therefore, novel agents are required. We established three TKI-resistant GIST patient-derived xenograft (PDX) models for effective drug development. These were PDX models harboring primary and secondary KIT and additional mutations; KIT exon 11 (p.Y570_L576del), KIT exon 17 (p.D816E), and PTEN (p.T321fs) mutations in GIST-RX1 from a patient who was unresponsive to imatinib, sunitinib, and sorafenib, and KIT exon 11 (p.K550_splice) and KIT exon 14 (p.T670I) mutations in GIST-RX2 and KIT exon 9 (p.502_503insYA) and KIT exon 17 (p.D820E) mutations in GIST-RX4 from patients with imatinib and imatinib/sunitinib resistance, respectively. The histological features and mutation statuses of GIST PDXs were consistent with those of the original patient tumors, and the models showed TKI sensitivity comparable to clinical responses. Imatinib inhibited the KIT pathway in imatinib-sensitive GIST-T1 but not GIST-RX1, RX2, and RX4. These GIST PDX models will be useful for studying TKI resistance mechanisms and evaluating novel targeted agents in GIST.

Abstract 4170: Targeting androgen receptor overcomes resistance to tyrosine kinase inhibitors in advanced clear cell renal cell carcinoma

Abstract Background: Advanced renal cell carcinoma (RCC) responds initially to anti-angiogenic therapies but eventually develop resistance which may be driven by the expression of key intratumoral pro-survival protein and overall kinome reprogramming. Androgen receptor (AR) expression has been reported in clear cell renal cell carcinoma (ccRCC) but its biological role remains elusive and its association with resistance to tyrosine kinase inhibitors (TKIs) has not been studied. Here we report the role of AR and its association with resistance to TKIs in advanced RCC. Methods: Human RCC cell lines; 786-0, 786-0R (with induced sunitinib resistance), ACHN, URMC2 (with intrinsic sunitinib resistance) were used to assess the combination effect of sunitinib and the AR antagonist enzalutamide in vitro. In vivo studies utilizing 786-0 tumors assessed the combination effect of enzalutamide and sunitinib following acquired resistance to sunitinib. AR expression was detected by qRT-PCR and Western blot analysis, and AR localization by immunofluorescence in treated and non-treated cells. Gene array was used to assess 93 AR associated genes in sensitive and resistant cells. Results: Quantitative RT-PCR data revealed a significant increase in AR expression with sunitinib resistance (&amp;gt;100 folds; p&amp;gt;0.001). In vitro and in vivo studies indicated significant increase in nuclear and cytoplasmic expression of AR with sunitinib treatment in resistant cells which was abrogated with single agent enzalutamide and combination treatment. More interestingly, combination treatment of enzalutamide and sunitinib significantly decreased cell growth and induced tumor regression in vitro and in vivo, respectively. Conclusion: Our data suggest the role of AR both in intrinsic and acquired sunitinib resistance in ccRCC and provide a rationale for combination strategies to restore TKI sensitivity that can be tested in the clinical setting. Citation Format: Remi M. Adelaiye-Ogala, Sreenivasulu Chintala, Ashley Orillion, May Elbanna, Ben Elzey, Nur Damayanti, Kiersten M. Miles, Chinghai Kao, Piergiorgio Pettazzoni, Giulio F. Draetta, Roberto Pili. Targeting androgen receptor overcomes resistance to tyrosine kinase inhibitors in advanced clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4170. doi:10.1158/1538-7445.AM2017-4170

Expanding the search for significant EGFR mutations in NSCLC outside of the tyrosine kinase domain with next-generation sequencing.

While conventional organization of EGFR mutations in non-small cell lung cancer (NSCLC) includes classic lesions sensitive to tyrosine kinase inhibitors (TKI) and variants localized to the tyrosine kinase domain (TKD) in exons 18-21, next-generation sequencing (NGS) raises the prospect of identifying clinically relevant variants in extra-TKD regulatory regions. NSCLC patients at our institution who received tumor profiling with NGS from 2013 to 2015 were identified. EGFR mutations were arranged based upon their distribution relative to the TKD. In silico analysis was performed to predict non-synonymous single nucleotide polymorphism (nsSNP) pathogenicity. Of 247 patients, 43 EGFR variants were seen in 39 patients (16%). While 32 had TKD lesions demonstrable through standard testing, 7 had extra-TKD nsSNPs (7/43), of which 5 were extracellular domain (ECD), 1 juxtamembrane (JM) and 1 carboxy-terminal (CT). Aside from known pathogenic ECD mutation G598V, 5/6 extra-TKD nsSNPs were predicted damaging with in silico analysis. Seven of 7 extra-TKD nsSNP+ patients smoked and were stage IV; 5/7 were adenocarcinoma. An adenocarcinoma patient with JM R675Q had erlotinib, 150mg daily, added following progression of disease on carboplatin and paclitaxel and had a partial response for 4months. No other extra-TKD nsSNP+ patient received EGFR-directed therapy. >2% NSCLC cases in our cohort had EGFR nsSNPs located outside of the TKD, representing >16% of all EGFR mutations. Extra-TKD variants should be characterized collaboratively to determine TKI sensitivity and additional therapeutic targets.

Expanding the search for significant EGFR mutations in NSCLC outside of the tyrosine kinase domain (TKD) with next-generation sequencing (NGS).

e20506 Background: While conventional organization of EGFR mutations in NSCLC includes classic lesions sensitive to tyrosine kinase inhibitors (TKI) and variants localized to TKD in exons 18-21, NGS raises the prospect of identifying clinically relevant variants in extra-TKD regulatory regions. Methods: Patients (pts) with lung cancer who received tumor profiling with NGS from 2013-2015 via Caris were identified. EGFR mutations were arranged based upon their known distribution relative to the TKD. In silico analysis was performed with PolyPhen-2 (Harvard) to predict nsSNPs’ pathogenicity. Results: 259 pts (248 NSCLC, 11 SC) had median age 65 years (26-85); 50% female; 64% white, 34% black; 73% with ≥20 pack-years (py), 12% non-smokers; 53% of samples were metastases. 65% NSCLC were adenocarcinoma (A), 21% squamous (S), 8% large-cell; 87% stage IV, 12% III. 44 EGFR variants were seen in 40 pts (15%; 39 NSCLC, 1 SC). While 32 pts had TKD lesions demonstrable through standard testing, 8 had extra-TKD mutations (8/44), of which 5 were extracellular domain (ECD), 1 juxtamembrane (JM) and 2 carboxy terminal (CT). Aside from pathogenic ECD mutation G598V, 5/7 extra-TKD nsSNPs were predicted-damaging (pnsSNP) with in silico (Table 1). 7/7 extra-TKD nsSNP+ pts smoked (6/7 ≥20 py) and all 6 NSCLC pts were stage IV; 50% A, 17% S; 83% male. The pt with JM R675Q had erlotinib, 150 mg daily, added following progression of stage IV NSCLC on carboplatin and paclitaxel and had a partial response for 4 months. No other pt received EGFR-directed therapy. Conclusions: 2% NSCLC cases in our cohort had EGFR pnsSNPs located outside of the TKD, representing &gt;18% of all EGFR mutations. Extra-TKD variants should be characterized collaboratively to determine TKI sensitivity and additional therapeutic targets. [Table: see text]

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.

Abstract PR02: Investigating the use of tyrosine kinase inhibitors in Ph-like ALL.

Abstract Ph-like or BCR-ABL1-like B-progenitor acute lymphoblastic leukemia (B-ALL) is characterized by a gene expression profile similar to BCR-ABL1 ALL, alteration of the lymphoid transcription factor IKZF1, and poor outcome. The prevalence of Ph-like ALL rises with age from 10% in standard risk childhood ALL to over 25% in young adults with ALL. We recently performed genomic profiling of over 1700 B-ALL cases and next-generation sequencing of over 150 Ph-like ALL cases and identified alterations targeting 18 kinase or cytokine receptor genes, including rearrangement, sequence mutation and copy number alterations. The aim of this study was to determine the role of these kinase alterations in the development of Ph-like B-ALL, to define the signaling pathways that are activated, and to assess sensitivity to tyrosine kinase inhibitors (TKIs). To investigate the functional role of kinase fusions in Ph-like ALL, we introduced RCSD1-ABL2, SSBP2-CSF1R or PAX5-JAK2 into interleukin-7 dependent Arf-/- mouse pre-B cells expressing the dominant negative isoform of IKAROS, IK6. ABL2 (Abelson-related gene, ARG), a homolog of ABL1, has been implicated in the development of solid tumors, but rarely identified as a rearrangement partner in ALL. CSF1R (encoding the macrophage colony stimulating receptor) regulates the survival and differentiation of macrophages, and is not normally expressed in lymphoid cells. Each fusion tested conferred cytokine independent growth in vitro. Furthermore, recipient mice transplanted with pre-B cells expressing RCSD1-ABL2 or SSBP2-CSF1R developed ALL with a pre-B immunophenotype (CD43+, B220+, CD19+, BP-1+ and IgM-) and a median latency of 36 and 40 days respectively, implicating these kinases for the first time in the development of B-ALL. To assess activation of kinase signaling pathways, we performed phosphoflow cytometry analysis of Arf-/- pre-B cells or human leukemic cells harvested from xenografted mice expressing ETV6-ABL1, RANBP2-ABL1, PAG1-ABL2, RCSD1-ABL2, SSBP2-CSF1R, IGH-EPOR, ATF7IP-JAK2 and PAX5-JAK2. In both models we observed distinct patterns of signaling pathway activation and TKI sensitivity between the different fusions. Cells expressing ABL1-class kinase fusions (ABL1, ABL2, CSF1R and PDGFRB) showed activation of pSTAT5 that was inhibited with imatinib or dasatinib. Phosphorylation of CRKL, a known target of ABL1 and ABL2, was only observed in cells expressing these fusions. In contrast, leukemic cells harboring ATF7IP-JAK2, PAX5-JAK2 or IGH-EPOR showed activation of pSTAT5 that was only attenuated with the JAK2 inhibitor, ruxolitinib. This TKI response profile was confirmed by cytotoxicity assays, with ABL-class fusions being sensitive to dasatinib (IC50 range 1-2nM), whilst cases with ATF7IP-JAK2 and EPOR rearrangement responded to ruxolitinib, with IC50 values of 500nM and 850nM respectively. To further examine the utility of TKI therapy in Ph-like B-ALL, we investigated the in vivo efficacy of dasatinib in four xenograft models of ETV6-ABL1, RCSD1-ABL2, PAG1-ABL2 or SSBP2-CSF1R ALL. Dasatinib (20mg/kg/day p.o) or vehicle were commenced at measurable engraftment in peripheral blood (human CD45+/CD19+ cells &amp;gt;5%) and treatment ceased when leukemic burden exceeded 70% in vehicle treated mice. In all cases, dasatinib significantly reduced the leukemic burden and splenic weight compared to vehicle treated mice. Furthermore, phosphorylation of STAT5 was attenuated in cells harvested from dasatinib-treated mice compared to vehicle, indicating on target effect of the drug. Together, these data provide the first mouse models of Ph-like B-ALL, and show that despite the diversity of alterations identified, they activate distinct signaling pathways that can be targeted with a limited panel of FDA-approved agents. Prospective clinical trials that identify and direct Ph-like ALL patients to the appropriate TKI therapy may improve the treatment outcome for this high-risk subtype. Citation Format: Kathryn G. Roberts, Yung-Li Yang, Debbie Payne-Turner, Richard C. Harvey, I-Ming Chen, Julie M. Gastier-Foster, Mignon L. Loh, Cheryl L. Willman, Stephen P. Hunger, Charles G. Mullighan. Investigating the use of tyrosine kinase inhibitors in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR02.

EGFR, KRAS, PIK3CA mutations and response to tyrosine kinase inhibitors (TKIs) in advanced NSCLC patients

22003 Background: Molecular alterations of the EGFR pathway are considered the major determinant of clinical outcome in response to EGFR TKIs in non small cell lung cancer (NSCLC) patients (pts).The aim of this study was to determine the clinical implication of EGFR, KRAS, PIK3CA gene mutations in pts with advanced NSCLC who had been treated with gefitinib or erlotinib after failure of 1st or 2st-line platinum-based chemotherapy. Methods: Genomic DNA was isolated from paraffin- embedded tumor specimens, amplified for EGFR (exons 18, 19, 20 and 21), PIK3CA (exons 9 and 20) KRAS (exon 2) by nested polymerase chain reaction and sequenced in both sense and antisense directions. RECIST criteria were used to assess response to TKIs. Results: From July 2002 to December 2007 128 pts have been treated with TKIs in our Institution. EGFR mutations were detected in 25.4% of pts; 16.4% had deletional mutations in exon 19, 4.7% point mutations in exon 20 and 4.7% in exon 21. KRAS and PIK3CA mutations were detected in 5.7% and in 2.7% of pts, respectively. Overall, pts with EGFR mutations had a response rate (RR) of 58.4% vs 19.7% (p=0.001), disease control rate of 74.2% vs 49.4% (p=0.02) median time to progression of 8 months vs 3 months (p=0.06), with respect to EGFR non mutated pts. No difference in overall median survival was observed. However when we considered 25 pts with TKI-sensitive EGFR mutations (deletion in exon 19, missense L858R) we observed a significant longer TTP (p=0.008, median 8 vs 3 months in non mutants) and a better OS (p=0.07, median not reached vs 11 months in non mutants). In this setting the RR was 88% (22 of 25) and TTP was significantly better also the multivariate analysis (p=0.03). All pts with KRAS mutations did not responded to TKIs treatment as the pts with PIK3CA mutations. Conclusions: In this experience we confirmed that EGFR mutations (deletion in exon 19, missense L858R) are the most important predictor of TKI sensitivity. In addition the evaluation of KRAS and PIK3CA mutations could contribute to identify lung cancer patients who are most suitable for TKIs treatment. No significant financial relationships to disclose.

The Need for an Individual Approach to Lung Cancer Treatment

Heidi Greulich and colleagues [ 1] have demonstrated the significance of insertion mutations in the epidermal growth factor receptor (EGFR) (exon 20) for tumorigenesis and responsiveness to tyrosine kinase inhibitors (TKIs) in lung cancers. They reported that different types of EGFR mutations conveyed different sensitivity to TKIs. Although several previous studies have suggested that tumors harboring mutations in the EGFR kinase domain were sensitive to TKIs such as gefitinib or erlotinib [ 2, 3], others had reported an association between the T790M mutation in exon 20 and the resistance to TKIs [ 4, 5]. As we learn more about the relationship between EGFR status (including gene copy number, mutation status, and mutation type) and drug sensitivity, decisions about treatment with TKIs for patients with non-small-cell lung cancer (NSCLC) become more complex. Previously, we reported HER2 mutational status, as well as EGFR and KRAS, in a large number of NSCLCs [ 6]. We found that 22% of tumors had EGFR kinase domain mutations (149 out of 671). Of these 149, 15 of them were insertion mutations in exon 20. The more common types of mutations—i.e., deletion in exon 19 (68 out of 149) and L858R in exon 21 (61 out of 149)—were more frequent in women and in “never smokers” (with p values of less than 0.001), whereas the exon 20 insertion mutations showed no bias for sex (seven in males versus eight in females) or smoking status (seven in smokers versus eight in never smokers). We have no data regarding TKI sensitivity for the 15 patients with insertion mutations to date, but based on the results from others and our own in vitro data, they may not benefit from the conventional TKIs, despite the fact that their tumors have EGFR kinase domain mutations. To maximize benefit to patients, we should determine the exact type of mutation for an individual tumor and determine whether it conveys sensitivity or resistance prior to TKI therapy. The development and clinical application of novel agents overcoming resistance should yield a more effective targeted therapy for tumors with all types of EGFR mutations.

Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers

Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene in lung cancers have generated enormous interest, because they predict for sensitivity to TK inhibitors (TKIs). While mutational status is of great importance in determining response to TKIs, it is not the sole factor, and evidence is accumulating that EGFR gene amplification, other members of the EGFR family (HER2, HER3) and genes downstream of EGFR signaling (KRAS, BRAF), may be involved in cancer pathogenesis and the response of TKIs. EGFR mutations occur in highly selected subpopulations of lung cancer patients: adenocarcinoma histology, never-smoker status, East Asian ethnicity and female gender. The recent finding of "a resistance associated" mutation for TKIs also provides new insights into this complicated mechanism. Thus, molecular-based studies to analyze the biological functions and to assess TKI sensitivity depending on the type of mutations are required. Epidemiological studies to identify possible carcinogenic factor(s) affecting different subpopulations are also of interest. In addition, for optimal therapeutic approach a comprehensive understanding of the genes related to EGFR signaling pathway, including RAS/RAF/MAPK and PI3K-AKT pathways, are required.

HER2 and HER3 genomic gain increases sensitivity to gefitinib in epidermal growth factor receptor positive advanced non-small cell lung cancer

3021 Background: In non-small cell lung cancer (NSCLC), sensitivity to tyrosine kinase inhibitors (TKIs) is associated with activating mutation and genomic gains of the epidermal growth factor receptor (EGFR). HER2 and HER3 overexpression increases sensitivity to TKI in pre-clinical settings. Mutations in the HER2 gene have recently been described, but their role in TKI sensitivity is unclear. Methods: One-hundred one NSCLC patients treated with gefitinib (250mg), and previously evaluated for EGFR status by fluorescence in situ hybridization (FISH) and DNA sequencing, were investigated for HER2 and HER3 genomic gains by FISH, and in frame insertions and duplications in HER2 exon 20 by DNA sequencing. Patients with high polysomy and gene amplification were considered as FISH positive (+). Results: FISH HER2+ and HER3+ patterns were significantly associated with female gender (p=0.03 and 0.02) and never smoking history (p=0.04 and 0.02). HER2+ patients (23%) had significantly better objective response (OR: 35% versus 6%, p=0.001), disease control rate (DCR: 56% versus 33%, p=0.04), time to progression (TTP: 9.05 versus 2.7 months, p=0.02), and a non significant trend toward longer survival (OS: 20.8 versus 8.4 months, p=0.056) than HER2-. EGFR+/HER2+ patients had a significantly better OR, DCR, TTP, and OS than patients EGFR-and/or HER2-. Patients EGFR+/HER2+ had better OR than EGFR+/HER2- (54% versus 21%, p=0.07), while EGFR-/HER2+ had same poor outcome than patients negative for both receptors. Patients HER3+ (26%) had a significant longer TTP (3.7 versus 2.7, p=0.04) than HER3-, but no better response or survival. Patients EGFR+/HER3+ had higher OR (36.4% versus 9.9%, p=0.03) and TTP (7.7 versus 2.7 months, p=0.03) than EGFR- and/or HER3-, while patients EGFR-/HER3- had lower response rate (18.2% versus 81.8%, p=0.02) and TTP (2.6 versus 6.0 months, p=0.002) than EGFR+ and/or HER3+. Mutation was not detected in exon 20 of the HER2 gene in 74 sequenced patients. Conclusions: HER2 and HER3 FISH analyses add to EGFR tests for prediction of clinical benefit to TKI therapy in advanced NSCLC patients. HER2 mutations seem to be infrequent and not clinically relevant. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Genentech, ImClone, Ligand, Roche AstraZeneca, Bristol-Myers Squibb, Genentech, ImClone, OSI, Roche