On-target Resistance Research Articles

EGFR-Mutated Lung Cancers Resistant to Osimertinib through EGFR C797S Respond to First-Generation Reversible EGFR Inhibitors but Eventually Acquire EGFR T790M/C797S in Preclinical Models and Clinical Samples

IntroductionOsimertinib is approved for advanced EGFR-mutated NSCLC, and identification of on-target mechanisms of resistance (i.e., EGFR C797S) to this third-generation EGFR inhibitor are evolving. Whether durable control of subsequently osimertinib-resistant NSCLC with the EGFR-sensitizing mutation (SM)/C797S is possible with first-generation EGFR inhibitors (such as gefitinib or erlotinib) remains underreported, as does the resultant acquired resistance profile. MethodsWe used N-ethyl-N-nitrosourea mutagenesis to determine the profile of EGFR SM/C797S preclinical models exposed to reversible EGFR inhibitors. In addition, we retrospectively probed a case of EGFR SM lung adenocarcinoma treated with first-line osimertinib, followed by second-line erlotinib in the setting of EGFR SM/C797S. ResultsUse of N-ethyl-N-nitrosourea mutagenesis against the background of EGFR L858R/C797S in conjunction with administration of gefitinib revealed preferential outgrowth of cells with EGFR L858R/T790M/C797S. A patient with EGFR delE746_T751insV NSCLC was treated with osimertinib with sustained response for 10 months before acquiring EGFR C797S. The patient was subsequently treated with erlotinib, with response for a period of 4 months, but disease progression ensued. Liquid biopsy disclosed EGFR delE746_T751insV with T790M and C797S present in cis. ConclusionEGFR SM NSCLC can acquire resistance to osimertinib through development of the EGFR C797S mutation. In this clinical scenario, the tumor may respond transiently to reversible first-generation EGFR inhibitors (gefitinib or erlotinib), but evolving mechanisms of on-target resistance—in clinical specimens and preclinical systems—indicate that EGFR C797S along with EGFR T790M can evolve. This report adds to the growing understanding of tumor evolution or adaptability to sequential EGFR inhibition and augments support for exploring combination therapies to delay or prevent on-target resistance.

Abstract 930: On-target resistance to mutant selective EGFR inhibitors develops in an allele-specific manner dependent on the original EGFR activating mutation

Abstract In-frame deletions within exon 19 (Ex19Del) and the Leu858Arg mutation within exon 21 (L858R) are the two most common oncogenic EGFR mutations found in non-small cell lung cancer (NSCLC). Currently, there is little data differentiating Ex19Del from L858R, and patients whose tumors harbor Ex19Del and L858R are approached similar in clinic when choosing EGFR tyrosine kinase inhibitors (TKIs). Unfortunately, resistance mutations may emerge against all classes of EGFR TKIs, including osimertinib. Recently, G724S has been identified as a novel mutation which is selected in osimertinib resistant tumors. Interestingly, in a large genomic profile database of NSCLC patient samples we found that G724S co-occurs preferentially with Ex19Del (15/19 G724S cases) but not with L858R (0/19 G724S cases). We further combined in silico and in vitro investigations to demonstrate the mechanism of G724S mutation confers resistance to TKIs in an oncogenic variant-dependent manner. Our in silico investigations showed evidence of reduced stability of the osimertinib-bound G724S/Ex19Del complex but not the osimertinib-bound G724S/L858R complex. G724S/Ex19Del-transduced cells are resistant to osimertinib treatment in vitro, while G724S/L858R-transduced cells are sensitive to osimertinib treatment. These results suggest G724S/Ex19Del may emerge under selective pressure from EGFR TKIs, and the underlying oncogenic profile of EGFR can influence the development of drug resistance mutations. Our results have potential direct implications for the treatment of patients with EGFR-mutant NSCLC. Citation Format: Yunkai Zhang, Benjamin P. Brown, David Westover, Yingjun Yan, Huan Qiao, Vincent Huang, Zhenfang Du, Jarrod A. Smith, Jeffrey S. Ross, Vincent A. Miller, Siraj Ali, Lyudmila Bazhenova, Alexa B. Schrock, Jens Meiler, Christine M. Lovly. On-target resistance to mutant selective EGFR inhibitors develops in an allele-specific manner dependent on the original EGFR activating mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 930.

Abstract 931: The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer

Abstract Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first generation TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations (C797S, G796S/R and L792F/H) that are mostly cytosine based. Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by digital droplet PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Knocking down AICDA by shRNA or CRISPR-Cas9, decreases the development of T790M in PC9 cell lines after TKI exposure. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFκB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFκB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFκB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFκB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, Luo Wang, April Davis, Alexander Cooke, Varun Vadnala, Hasan Korkaya, Gregory P. Kalemkerian, Khaled A. Hassan. The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 931.

Abstract 929: Paired tumor and cfDNA in patients with HER2-mutant solid tumors treated with neratinib reveals convergence of multiple on-target resistance mechanisms: Results from the SUMMIT "Basket" Trial

Abstract Background: Somatic mutations in ERBB2 occur across various tumor types at relatively low frequencies and can lead to constitutive kinase signaling and oncogenic transformation. SUMMIT is an ongoing basket trial of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, in pts with ERBB2 mutant cancers (NCT01953926). Methods: Pts with advanced solid tumors and locally documented ERBB2 mutations received neratinib 240 mg daily (N=8) +/- fulvestrant at the labeled dose for pts with ER+ breast cancer (N=6) as part of the global Phase II SUMMIT basket study. Pre- and post-treatment tumor and cfDNA were subjected to NGS using MSK-IMPACT for tissue (410 genes) and MSK-ACCESS for plasma (129 genes) to identify SNVs, indels, and copy number alterations. Results: In total, 14 pts had successful sequencing of both paired tissue and cfDNA sample (10 breast, 2 gallbaldder, 1 bladder, and 1 unknown primary). All pts achieved clinical benefit on neratinib including 2 CRs, 7 PRs, and 5 SDs. In 1 gallbladder pt, paired tissue sequencing identified loss of the pretreatment clonal ERBB2 mutation but retention of a truncal TP53 mutation, suggesting emergence of a distantly related ERBB2 wildtype clone. In the remaining 13 patients, the pretreatment ERBB2 mutation was retained in tissue at progression. 64% (9/14) pts had at ≥1 acquired alteration in tissue including 3 pts who acquired secondary alterations in ERBB2, both clonal and subclonal ERBB2 mutations as well new focal amplifications. Consistent with this finding, plasma cfDNA sequencing revealed 57% (8/14) of pts acquired ≥1 secondary ERBB2 mutation with the majority occurring at known activating hotspots. Analysis of mutant allele frequencies of these emergent ERBB2 alterations, in comparison to other variants, suggested the majority were subclonal with evidence of multiple independent subclones arising in the same patient. Two pts developed known ERBB2 gatekeeper mutations (T798I and L785F). Conclusion: In pts with ERBB2-mutant solid tumors with clinical benefit on neratinib, a potential on-target resistance mechanism was identified in 71% (10/14, including 7 with acquired gain-of-function ERBB2 mutations, 2 with ERBB2 both gain-of-function and gatekeeper mutations, and 1 with outgrowth of an ERBB2 wildtype clone). Collectively, these data provide additional evidence that ERBB2 mutations lead to oncogene addiction in solid tumors. We also demonstrate that tumor and cfDNA sequencing provides complementary information that can be integrated to more fully elucidate potential resistance mechanisms. Citation Format: Helen H. Won, S. Duygu Selcuklu, Sarina A. Piha-Paul, Cristina Saura, Jordi Rodon, Ingrid A. Mayer, Sherene Loi, Geoffrey I. Shapiro, Janice Lu, Adam Brufsky, Catherine Zimel, Myra Melcer, Maurizio Scaltriti, Lisa D. Eli, Richard E. Cutler Jr., Alshad S. Lalani, Richard P. Bryce, Carlos Arteaga, Funda Meric-Bernstam, Michael F. Berger, David B. Solit, Alison Schram, David M. Hyman. Paired tumor and cfDNA in patients with HER2-mutant solid tumors treated with neratinib reveals convergence of multiple on-target resistance mechanisms: Results from the SUMMIT "Basket" Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 929.

Abstract 442: Repotrectinib, a next generation TRK inhibitor, overcomes TRK resistance mutations including solvent front, gatekeeper and compound mutations

Abstract Oncogenic TRKA/B/C fusions are identified in multiple cancer types in adults and children. TRK inhibitors (TRKis) have demonstrated marked efficacy in patients with these cancers, however, acquired on-target resistance mediated by kinase domain mutations can occur. Next-generation TRKis targeting both wildtype and mutant TRK fusions can address this unmet need. Repotrectinib was designed to potently inhibit wildtype (WT) TRKs and overcome resistance mutations. The anti-proliferative activity of 1st generation (larotrectinib/entrectinib) and next-generation (repotrectinib/LOXO-195) TRKis were compared using engineered Ba/F3 cells expressing WT or mutated TRKs (Table). Repotrectinib was over 10-fold more potent than LOXO-195 against WT TRK fusions and solvent front mutations (SFMs), and more than 100-fold more potent against the gatekeeper mutations TRKA F589L and TRKC F617I. Furthermore, repotrectinib was the only TRKi active against the compound mutation TRKA G595R/F589L in cis in preclinical Ba/F3 cells. In xenograft tumor models, repotrectinib led to significant tumor regression in tumors carrying WT or mutated TRK fusions. In the ongoing TRIDENT-1 phase 1 clinical trial of repotrectinib (NCT03093116), the SFMs TRKA G595R, TRKC G623R and TRKC G623E and the gatekeeper mutation TRKA F589L were detected in plasma cfDNA samples at baseline from three TRKi-resistant patients. Repotrectinib was active against ETV6-TRKC G623E in an entrectinib-resistant patient with a salivary gland tumor (-82%, confirmed partial response, RECIST v1.1). Tumor regression (-33%) was achieved in a larotrectinib-resistant cholangiocarcinoma patient with LMNA-TRKA G595R and F589L mutations in trans. TRIDENT-1 is currently enrolling NTRK fusion-positive patients with advanced solid tumors. Ba/F3 Cell Proliferation Assay IC50 (nM)LMNA-TRKAETV6-TRKBETV6-TRKCTRK InhibitorWTG595RG667CF589LG595R/F589LWTG639RWTG623RG623EF617IRepotrectinib<0.10.19.2<0.117.4<0.13.2<0.10.40.9<0.2LOXO-1958.613.194.931.6531.11.028.41.724.649.153Larotrectinib15.918981863625.31000013.1300023.2699921625000Entrectinib0.3614186.7<0.220000.220000.41340171261.7 Citation Format: Alexander Drilon, Dayong Zhai, Wei Deng, Xin Zhang, Dong Lee, Evan Rogers, Jeffrey Whitten, Zhongdong Huang, Armin Graber, Juliet Liu, Shanna Stopatschinskaja, J. Jean Cui, Dong-Wan Kim, Byoung Chul Cho, Robert C. Doebele, Sai-Hong Ignatius Ou, Jeeyun Lee, Alice T. Shaw. Repotrectinib, a next generation TRK inhibitor, overcomes TRK resistance mutations including solvent front, gatekeeper and compound mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 442.

Tissue-based molecular and histological landscape of acquired resistance to osimertinib given initially or at relapse in patients with EGFR-mutant lung cancers.

9028 Background: Even though osimertinib (osi) is now the initial treatment for patients with EGFR-mutant lung cancers, our knowledge about mechanisms of resistance (MOR) is largely derived from patients who received osi after developing acquired resistance to initial EGFR inhibitor. Further, studies of osi resistance to date have mainly reported genotyping of plasma which suboptimally detects lineage plasticity, copy number changes, and chromosomal rearrangements. Methods: To identify MOR to osi and characterize clinical, molecular and histologic factors associated with duration of response, we identified patients with EGFR-mutant lung cancers who had next-generation sequencing performed on tumor tissue after developing acquired resistance to osi. Results: From January 2016 to December 2018, post-osi tumor tissue was collected from 71 patients (42 with paired pre-treatment specimens). See mechanisms of resistance below. Histologic transformation was identified in 19% of initial cases and 14% of all cases. When osi is given as initial treatment, with median follow up of 17 months, early emerging MOR rarely included on-target resistance mechanisms (1/16 cases of acquired EGFR G724S). Acquired alterations representing potential resistance mechanisms included CCNE1 and MYC amplifications, and mutations in MTOR and MET H1094Y. We confirmed in preclinical studies that an amino acid substitution at MET H1094 can reduce sensitivity to osi. Conclusions: In this analysis of MOR identified on NGS from tumor tissue, we found a different spectrum of resistance mechanisms to initial and later-line osi, with histologic transformation (including squamous cell transformation) a dominant MOR, particularly in the first-line setting, that cannot be identified on plasma testing. Subsequent studies are needed to assess patients with a longer time on initial osi as there may be a temporal bias to MOR, with off-target MOR emerging earlier and on-target resistance mutations later. [Table: see text]

TRK xDFG Mutations Trigger a Sensitivity Switch from Type I to II Kinase Inhibitors

On-target resistance to next-generation kinase inhibitors, drugs specifically designed to maintain potency in the setting of kinase domain mutations, remains poorly characterized. Having identified that xDFG (TRKA G667) mutations confer second-generation TRK inhibitor resistance in patients with TRK fusion-positive cancers, we used computational modeling and biochemical assays to study how these affect drug binding. We found that xDFG mutations stabilize the inactive kinase conformation, hampering the binding of type I second-generation TRK inhibitors, while increasing the affinity for type II multikinase inhibitors. Type II drugs consistently inhibit the growth and TRK-mediated signaling of isogenic and patient-derived models harboring xDFG mutations. Collectively these data demonstrate that TRK xDFG substitutions trigger conformational changes that favor type II binding mechanisms. Given prior identification of paralogous xDFG resistance mutations in other oncogene addicted cancers, these findings provide insight into rational drug design to address these recalcitrant resistant alterations by leveraging inhibitor class affinity switching.

Abstract 5836: Activation Induced Cytosine Deamination, AICDA, is induced after EGFR TKI exposure leading to secondary resistant mutations in lung adenocarcinoma

Abstract Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first line TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations, C797S, G796S/R and L792F/H. Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by droplet digital PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Knocking down AICDA by shRNA, decreases the development of T790M in PC9 cell lines after TKI exposure. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFĸB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFĸB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFĸB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFĸB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, April Davis, Alexander Cooke, Luo Wang, Hasan Korkaya, Gregory Kalemkerian, Khaled Hassan. Activation Induced Cytosine Deamination, AICDA, is induced after EGFR TKI exposure leading to secondary resistant mutations in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5836.

Abstract 936: Analysis of cell-free DNA from 32,991 advanced cancers reveals novel co-occurring activating RET alterations and oncogenic signaling pathway aberrations

Abstract Background The receptor tyrosine kinase (RTK), RET, is an emerging oncogenic target. Inhibitors targeting activating RET alterations (alts), including on-target resistance alts, are being studied in solid tumors. We present a large cohort of patients (pts) with metastatic solid tumors and somatic RET alts detected by next generation sequencing (NGS) of cell-free DNA (cfDNA). cfDNA provides a global description of RET and other genomic alts in advanced disease, typically exposed to one or more lines of systemic therapy, complementing previous descriptions of RET alts in tissue which is often obtained from early stage/untreated tumors (e.g. TCGA). Methods Somatic activating RET alts (fusions/missense alts predicted to be oncogenic) were identified from 32,991 consecutive pts tested with a 68-73 gene cfDNA assay (Guardant Health, CA) between 02/2015-07/2017. This comprehensive NGS assay evaluates single nucleotide variants, and select indels, fusions, and copy number gains. Variants of uncertain significance and synonymous alts were excluded. Results 175 somatic activating RET alts were detected in 170 pts with non-small cell lung (NSCLC, n=123), colorectal (n=15), breast (n=8), thyroid (n=8), or other cancers (n=13). 141 pts had 142 RET fusions involving 7 different partners, most commonly KIF5B (n=77). Nineteen unique activating RET missense alts were detected in 29 pts, the most common being M918T (n=8). Two pts had multiple activating RET missense alts. Advanced cancers with activating RET alts commonly harbored additional alts in oncogenic signaling pathway genes: other RTKs (n=57 pts), cell cycle (n=48), MAPK (n=46), PI3K (n=27). The frequency and distribution of alts in these co-occurring pathway genes varied by specific RET alt. Missense RET alts had a lower proportion of co-occurring TP53 alts and a higher proportion of PIK3CA alts compared to RET fusions. Non-KIF5B-RET fusions were enriched for co-occurring alts in MAPK and other RTK genes relative to tumors with KIF5B-RET fusions. In pts with NSCLC, KIF5B-RET fusions were rarely observed with other bona fide oncogenic drivers whereas known EGFR driver alts were identified in pts with CCDC6-RET (10/31), NCOA4-RET (4/14), and TRIM24-RET (1/2) fusions. 12/15 had EGFR T790M and 3/12 had C797S mutations. Prior treatment with anti-EGFR therapy was confirmed in 5 pts. The RET fusion arose without (n=1), concurrent with (n=2), and subsequent to (n=2) emergence of T790M. Conclusions In the largest cohort of pts with advanced cancers harboring RET activating alts, we describe novel co-occurrences of oncogenic signaling pathway aberrations. Additionally, our findings suggest that CCDC6- and NCOA4-RET fusions may contribute to anti-EGFR therapy resistance in NSCLC. Understanding the clinical significance of co-occurring alts is essential to evaluating the efficacy of RET-directed therapies. Citation Format: Karen L. Reckamp, Thereasa A. Rich, Young Kwang Chae, Robert C. Doebele, Wade T. Iams, Michael Oh, Victoria M. Raymond, Richard B. Lanman, Thomas E. Stinchcombe, Vivek Subbiah, David R. Trevarthen, Oliver P. Gautschi. Analysis of cell-free DNA from 32,991 advanced cancers reveals novel co-occurring activating RET alterations and oncogenic signaling pathway aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 936.

Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound ALK Mutations in ALK-Positive Lung Cancer.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, N-ethyl-N-nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4-ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies.Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714-29. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Overcoming On-Target Resistance to Tyrosine Kinase Inhibitors in Lung Cancer

Advances in genomics, an improved understanding of malignant transformation, and the development of potent small molecule inhibitors capable of targeting key kinases have led to the adoption of genotype-guided approaches for the treatment of advanced cancers. As regulators of complex signaling networks, tyrosine kinases are among the most attractive targets. Moreover, insight into the conserved three-dimensional structures of these kinases and their mechanism of activation has facilitated the development of selective tyrosine kinase inhibitors (TKIs). TKIs have shown robust clinical activity in many different oncogene-addicted cancers; however, resistance invariably develops. In a significant proportion of patients, resistance results from acquired genetic alterations within the kinase target that allow cancer cells to escape TKI-mediated growth suppression. In this review, we discuss clinically observed and preclinical on-target resistance events in oncogene-driven solid tumors and describe current and future therapeutic strategies to overcome this type of resistance.

Recurrent Mutations in CCND3 Confer Clinical Resistance to FLT3 Inhibitors

Background. Activating mutations in FLT3 occur in ~30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (~25%) and point mutations in the tyrosine kinase domain (KD). In recent years, multiple selective and potent FLT3 inhibitors such as quizartinib, PLX3397, crenolanib and ASP2215 have demonstrated encouraging preliminary clinical activity in FLT3 mutant AML. Both quizartinib (Schiller et al, ASCO 2014) and ASP2215 (Levis et al, ASCO 2015) have reported composite complete remission rates (CRc) as high as ~50% in phase II trials. Despite these high response rates, the majority of patients relapse after initial response (acquired resistance) and a significant proportion of patients also fail to respond at all (primary resistance). On-target resistance due to secondary KD mutations in FLT3 is a common cause of acquired resistance to the clinically active FLT3 inhibitors quizartinib (Smith et al. Nature 2012) and PLX3397 (Smith et al, Cancer Discovery 2015). However, the causes of primary clinical resistance to FLT3 inhibitors have not been characterized. We performed targeted capture based sequencing of sorted pre-treatment blasts from 8 responding (R) and 21 non-responding (NR) patients treated on the phase I/II trial of PLX3397 in FLT3-ITD+ AML. We deeply sequenced the coding regions of 585 genes known to be mutated in hematologic malignancies/solid tumors and identified genes mutated only in NR patients (compared to genes mutated in both R and NR patients).Results. The number of mutations detected in genes other than FLT3 ranged from 2-18 per sample. There was no increase in the number of mutations found in patients with pre-existing hematologic conditions or in NR patients. Surprisingly, one of the most frequently mutated genes observed exclusively in NR patients was CCND3, the gene encoding cyclin D3, which has rarely been reported to be mutated in AML, though it is mutated in 38% of sporadic Burkitt's lymphoma (BL). A total of 4 individual mutations in CCND3 (Q276*, Q280fs, R271fs, and T283A) were identified in 3/21 NR patients (one patient had both Q276* and Q280fs). No CCND3 mutations were found in R patients. The identified mutations were the same mutations commonly found in BL, known to result in a more stable isoform of cyclin D3 and retain sensitivity to CDK4/6 inhibitors (Schmitz et al., Nature 2012). Expression of the Q276* and T283A mutations in FLT3-ITD+ MV4;11 cells conferred resistance to apoptosis induced by several FLT3 inhibitors (PLX3397, AC220 and crenolanib). However, inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells by either the CDK4/6 inhibitor palbociclib or the combined FLT3-CDK4/6 inhibitor AMG925 (FLX925) was unable to restore sensitivity to FLT3 inhibition. Moreover, CCND3 mutant MV4;11 cells demonstrated no increase in Rb phosphorylation, suggesting resistance to FLT3 inhibitors facilitated by CCND3 mutations is not predicated on CDK4/6 activation of Rb-dependent E2F-mediated transcription.Conclusions. We have identified recurrent mutations in CCND3, a gene not previously known to be commonly mutated in AML, as a novel cause of clinical primary resistance to FLT3 inhibitors in AML. This represents the first report of a specific non-FLT3 dependent mechanism of clinical resistance to FLT3 inhibitors. Unlike in BL, the functional effects of CCND3 mutations in FLT3-ITD+ AML do not appear to be mediated via CDK4/6 activity and is therefore unresponsive to CDK4/6 inhibition. The molecular mechanism(s) of CCND3-mediated resistance to FLT3 inhibitors are currently being investigated. DisclosuresSmith:Plexxikon: Research Funding; Astellas: Research Funding. Off Label Use: Investigational use of PLX3397 in AML. Hsu:Plexxikon Inc.: Employment. West:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Shah:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Plexxikon Inc.: Research Funding.

Identification of TKI-Sensitive Point Mutations That Activate c-ABL Kinase Activity and Transformation Potential and Confer in Vitro Resistance to the Allosteric ABL Inhibitor GNF-5

Introduction:Pathologically activated tyrosine kinases represent attractive therapeutic targets, with deregulated ABL kinase representing one of the best-validated examples.While physiologic activation of c-ABL is tightly controlled by intramolecular interactions, ABL fusion proteins are constitutively activated by oligomerization driven by the fusion partner, as well as by loss of kinase autoinhibition due to removal of an autoinhibitory myristate site at the N-terminus of c-ABL (Nagar et al, Cell, 2003). Recent routine genomic tumor sequencing of various human malignancies has identified point mutations of unknown significance in numerous kinases, including c-ABL, and it is expected that the increasingly common practice of tumor genome sequencing will provide new opportunities for tailored targeted therapy. To maximize therapeutic benefit and avoid unnecessary cost and toxicity, it will be critical to distinguish "driver" from "passenger" mutations. We therefore sought to: (i) test the transforming potential of select clinically detected c-ABL mutants, (ii) prospectively identify novel additional activating c-ABL point mutations, and (iii) determine if active mutant c-ABL isoforms retain sensitivity to ABL TKIs.Results:Two of four clinically identified c-ABL mutations tested caused a dramatic increase in ABL kinase activity and transformed Ba/F3 cells to growth factor independence. A mutagenesis screen of c-ABL identified six additional transforming point mutations in the SH3 and kinase domains, including one that has been reported clinically in breast invasive carcinoma (cBioPortal.org). Relative to BCR-ABL, all c-ABL mutants showed heightened sensitivity to imatinib. Allosteric site inhibitors targeting the myristate-binding pocket in the BCR-ABL kinase domain (KD) have been shown to inhibit BCR-ABL kinase activity, purportedly by mimicking binding of the c-ABL myristate group and establishing c-ABL-like autoinhibitory constraints (Hantschel, Haematologica, 2012). While only two c-ABL mutations from the screen were near the allosteric site, all mutants exhibited decreased sensitivity to the allosteric inhibitor GNF-5. Recent studies have shown that derivative compounds of GNF-5 targeting this site in BCR-ABL are subvertable by on-target resistance mutations. We tested whether three BCR-ABL mutations described to confer in vitro, and in one case clinical, resistance to ABL001 (currently undergoing clinical trial evaluation) could impact c-ABL kinase activity presumably by perturbing this autoinhibitory pocket. In the context of c-ABL, all three ABL001-resistant mutations activated ABL kinase, transformed Ba/F3 cells, and conferred substantial resistance to allosteric inhibition relative to native BCR-ABL. These findings raise the possibility that c-ABL mutations may play a role in resistance to emerging allosteric ABL inhibitors. Importantly, previous work suggested that the genomic breakpoint on chromosome 9 in one third of CML cases is located 5' to ABL exon 1b, which would be predicted to result in simultaneous KD mutation expression in BCR-ABL and c-ABL (Uphoff et al, Leuk Res, 1999). To definitively demonstrate that co-expression of KD mutations in c-ABL and BCR-ABL can occur in primary patient samples, we analyzed RNA from five CML patients harboring a dominant BCR-ABL T315I mutation, and identified a heterozygous c-ABL 1b T315I mutation in one case.Conclusions:We have identified eight novel activating point mutations in c-ABL (three found in clinical isolates) that are potential therapeutic targets of ABL TKIs. Activating c-ABL mutants, including T315I, confer substantial resistance to the allosteric ABL inhibitor GNF-5 regardless of proximity to the inhibitor-binding site, implying resistance mechanisms beyond mere steric hindrance. Thus, mutations may induce distal conformational changes by disrupting a kinase conformation required for allosteric compound inhibition. In a subset of CML cases, c-ABL point mutants are co-expressed from the same allele as BCR-ABL, suggesting that the location of the chromosome 9 breakpoint may negatively impact clinical responsiveness to allosteric ABL inhibitors. Sequence analysis of c-ABL in patients with resistance to allosteric ABL inhibitors is required to validate c-ABL point mutants as a clinically important mechanism of resistance to this emerging class of targeted therapeutics. DisclosuresOff Label Use: the use of ABL TKIs for malignancies associated with point mutations in c-ABL. Shah:Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Plexxikon Inc.: Research Funding.

Computational metallomics of the anticancer drug cisplatin

Cisplatin, cis-diamminedichlorido-platinum(II), is an important therapeutic tool in the struggle against different tumors, yet it is plagued with the emergence of resistance mechanisms after repeated administrations. This hampers greatly its efficacy. Overcoming resistance problems requires first and foremost an integrated and systematic understanding of the structural determinants and molecular recognition processes involving the drug and its cellular targets. Here we review a strategy that we have followed for the last few years, based on the combination of modern tools from computational chemistry with experimental biophysical methods. Using hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) simulations, validated by spectroscopic experiments (including NMR, and CD), we have worked out for the first time at atomic level the structural determinants in solution of platinated cellular substrates. These include the copper homeostasis proteins Ctr1, Atox1, and ATP7A. All of these proteins have been suggested to influence the pre-target resistance mechanisms. Furthermore, coupling hybrid QM/MM simulations with classical Molecular Dynamics (MD) and free energy calculations, based on force field parameters refined by the so-called “Force Matching” procedure, we have characterized the structural modifications and the free energy landscape associated with the recognition between platinated DNA and the protein HMGB1, belonging to the chromosomal high-mobility group proteins HMGB that inhibit the repair of platinated DNA. This may alleviate issues relative to on-target resistance process. The elucidation of the mechanisms by which tumors are sensitive or refractory to cisplatin may lead to the discovery of prognostic biomarkers. The approach reviewed here could be straightforwardly extended to other metal-based drugs.

Targeting cancer with kinase inhibitors.

Kinase inhibitors have played an increasingly prominent role in the treatment of cancer and other diseases. Currently, more than 25 oncology drugs that target kinases have been approved, and numerous additional therapeutics are in various stages of clinical evaluation. In this Review, we provide an in-depth analysis of activation mechanisms for kinases in cancer, highlight recent successes in drug discovery, and demonstrate the clinical impact of selective kinase inhibitors. We also describe the substantial progress that has been made in designing next-generation inhibitors to circumvent on-target resistance mechanisms, as well as ongoing strategies for combining kinase inhibitors in the clinic. Last, there are numerous prospects for the discovery of novel kinase targets, and we explore cancer immunotherapy as a new and promising research area for studying kinase biology.

Mutations In UBA3 Confer Resistance To The NEDD8-Activating Enzyme Inhibitor MLN4924 In Human Leukemic Cells

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational conjugation of NEDD8 onto target proteins. Cullin proteins are major substrates of the neddylation pathway, and are involved in diverse cellular processes. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924 in leukemic cells.MLN4924 shows potent activity in K562 human leukemia cells (EC50 = 100 nM). In contrast, by exposing K562 cells to increasing concentrations of MLN4924 over 6 months, we selected a population of cells resistant to MLN4924 (R-K562MLN; EC50 > 2 µM). R-K562MLN cells proliferated similarly to parental K562 cells, and remained resistant to MLN4924 even after culturing in the absence of MLN4924 for 5 weeks. Compared to parental cells, R-K562MLN cells were similarly sensitive to a broad spectrum of chemotherapeutic drugs, including daunorubicin, a classical substrate of the multidrug efflux transporter P-glycoprotein. Thus, the mechanism of resistance to MLN4924 did not appear to be related to generalized multidrug efflux. While basal levels of NEDD8-conjugated cullin proteins were equivalent in parental and resistant cells, NEDD8-cullins were diminished in parental cells treated with 25 nM MLN4924. In contrast, no appreciable reduction in NEDD8-cullins was observed in R-K562MLN cells after treatment with up to 250 nM MLN4924. However, knockdown of NEDD8 by shRNA was cytotoxic to R-K562MLNcells, suggesting that NEDD8 conjugation remains essential for the survival of resistant cells.To further probe mechanisms of MLN4924 resistance in these cells, we sequenced the coding region of the NAE catalytic subunit UBA3 from K562 and R-K562MLN cells, and identified a mutation in codon 310 [ATT (Isoleucine, I) > AAT (Asparagine, N)] in R-K562MLN cells. Biochemical analyses using recombinant wild-type and I310N NAE complexes indicated that the I310N mutation alters biochemical properties of the NAE, increasing the enzyme's affinity for ATP while decreasing its affinity for NEDD8. The I310N NAE complex was ∼7-fold less sensitive to MLN4924 (IC50 = 225 nM) compared to wild-type NAE (IC50 = 32 nM), providing a mechanistic basis for MLN4924 resistance in R-K562MLNcells through the UBA3 I310N mutation.To investigate whether another malignant leukemic cell line employs a similar on-target resistance mechanism, we similarly selected a population of U937 cells resistant to MLN4924 (R-U937MLN). Parental U937 cells were sensitive to MLN4924 (EC50 = 25 nM), while R-U937MLN cells were more than 40-fold more resistant (EC50 > 1 µM). Sequencing of the coding region of UBA3 in U937 and R-U937MLN cells revealed a point mutation in codon 352 [TAT (Tyrosine, Y) > CAT (Histidine, H)] only in R-U937MLN cells. As for the UBA3 I301N mutation, the Y352H mutation conferred a ∼10-fold decrease in the sensitivity of the NAE to MLN4924 (IC50= 138.5 nM and 13 nM for Y352H and wild-type, respectively) through decreased NEDD8 and increased ATP affinities. These findings suggest that the Y352H mutation, like I310N, is sufficient to provide MLN4924 resistance in leukemia cells while sustaining adequate NAE function for leukemic cell survival.We next found that R-K562MLN cells were cross-resistant to other NAE-selective inhibitors derived from Compound 1, a pan-E1 inhibitor. Nevertheless, compared to parental cells, R-K562MLN cells were not refractory to Compound 1 (EC50 = 27 nM and 81 nM, respectively). The cytotoxicity of Compound 1 towards MLN4924-resistant cells might be explained by its inhibition of other E1 enzymes. Indeed, Compound 1 diminished the abundance of ubiquitinated proteins in R-K562MLNcells similarly to its effects in K562 cells, while NEDD8-cullins in the MLN4924-resistant cells were unaffected.Overall, as MLN4924 continues to be evaluated clinically, our work provides insight into the mechanisms of MLN4924 resistance, which may facilitate the development of more effective second-generation NAE inhibitors. Disclosures:Petroski:Allostere, Inc.: Consultancy, Equity Ownership.

Molecular mechanisms of cisplatin resistance

Platinum-based drugs, and in particular cis-diamminedichloroplatinum(II) (best known as cisplatin), are employed for the treatment of a wide array of solid malignancies, including testicular, ovarian, head and neck, colorectal, bladder and lung cancers. Cisplatin exerts anticancer effects via multiple mechanisms, yet its most prominent (and best understood) mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of mitochondrial apoptosis. Despite a consistent rate of initial responses, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. An intense research has been conducted during the past 30 years and several mechanisms that account for the cisplatin-resistant phenotype of tumor cells have been described. Here, we provide a systematic discussion of these mechanism by classifying them in alterations (1) that involve steps preceding the binding of cisplatin to DNA (pre-target resistance), (2) that directly relate to DNA-cisplatin adducts (on-target resistance), (3) concerning the lethal signaling pathway(s) elicited by cisplatin-mediated DNA damage (post-target resistance) and (4) affecting molecular circuitries that do not present obvious links with cisplatin-elicited signals (off-target resistance). As in some clinical settings cisplatin constitutes the major therapeutic option, the development of chemosensitization strategies constitute a goal with important clinical implications.

Abstract B18: Variation in response to VEGFR inhibitor tivozanib in a unique population-based tumor model enables the development of a multigene response biomarker

Abstract Variation is a hallmark of human tumors. Within any human tumor population, significant inter-tumor variation in genetic context underlies variation in phenotype, prognosis, and response to a given therapeutic agent. The ability to identify associations between genetic context and drug response lies at the core of much of translational research in cancer. However, current preclinical models fail to adequately represent this variation in a manner suitable for correlating genetic context with drug response. To address this challenge, we used complex genetically engineered murine tumors to develop a population based model comprising over 100 HER2 INK4A-/- driven breast tumors, each with associated expression microarray, array CGH, histology, as well as protein and biochemical characteristics. Similar to that observed in human tumor populations, this breast tumor archive exhibited significant inter-tumor variation in RNA and DNA profiles, and variation in many measurable tumor phenotypes, including tumor vasculature. The VEGF/VEGFR axis is postulated to be the dominant human tumor angiogenesis mediator. However, in contrast to robust activities observed in traditional xenograft models, anti-VEGF/VEGFR agents have thus far elicited relatively modest activity in human clinical studies (e.g. single agent RECIST response rates vary between 0–11% in several breast cancer studies). To better model the observed human variation in response to VEGF pathway antagonism, and to explore the development of a predictive biomarker for patient selection, we determined the responsiveness of tumors across the population based model to a potent, selective VEGFR inhibitor, tivozanib (AV-951). Tivozanib exhibits picomolar inhibitory activity against all three VEGF receptors, is active in a broad array of traditional xenografts, exhibits a multiday T1/2 in humans, and demonstrates robust clinical activity in renal cell carcinoma, with RECIST response rates of 25–40%. Interestingly, a significant variation in response to tivozanib was observed among 25 tumors in the population model, with the majority exhibiting intrinsic resistance to tivozanib's anti-angiogenic activity. Combining the efficacy data, extensive IHC analyses, the comprehensive expression profiles and a novel bioinformatics approach, we identified a multigene biomarker of on-target drug resistance. Using the same multigene biomarker identified in the murine model to examine human tumor microarray datasets, subsets of breast, lung, colon and kidney cancer human populations were predicted to exhibit tivozanib resistance. These data demonstrate the promise of population based preclinical models for translational research and provide a readily testable multigene biomarker for the potent VEGF pathway antagonist tivozanib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B18.

Anticipating drug resistance in the MAP kinase pathway

Anticipating drug resistance in the MAP kinase pathway