Additional Resistance Mechanisms Research Articles

Abstract B27: Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines

Abstract Oral squamous cell carcinoma (OSCC) remains a disease with poor survival. With recent characterization of the mutational landscape of OSCCs, there is great potential for personalized targeted therapies. However, to date utilization of targeted therapies in OSCC have had limited success with anti-EGFR therapy. Combinations of targeted therapies, which may have greater efficacy by inhibiting compensatory pathways, have not been well studied. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing, and by using a pool of CRISPRs targeting over 18,000 genes we can perform genome-scale screening for synergistic targetable pathways. Upon selection of the OSCC GeCKO pool, we identified that knockouts of genes in the FGFR pathway caused sensitivity to the EGFR inhibitor gefitinib. We further investigated the possible synergistic effect of dual EGFR and FGFR inhibition with resazurin viability assays in 14 of our OSCC models. We found 6/14 (43%) of the models were responsive to the combination of EGFR and FGFR inhibition, indicating that the FGFR pathway could be an alternate mechanism of resistance to EGFR-targeted therapy. For further investigation of this combination, we again used CRISPR-Cas9 to generate an EGFR knockout (KO) OSCC cell line. This EGFR KO cell line retains sensitivity to FGFR inhibitors, and we expect that this cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway as a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Hui Jiang, Thomas Carey, John Chad Brenner. Identifying therapeutic combinations with EGFR-targeted therapy through the generation of genome-wide CRISPR-Cas9 knockout libraries in oral cancer cell lines [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B27.

MOLECULAR BACKGROUND OF FLUOROQUINOLONE RESISTANCE IN PATHOGENIC HUMAN MYCOPLASMAS

Human mycoplasma pathogens are resistant to many types of antibiotics because of the lack of a cell wall. Macrolides are most often used for the treatment of mycoplasmosis, but the spread of forms resistant to these antibiotics requires the use of alternative treatment regimens, in particular, the administration of f luoroquinolones and tetracyclines. Of the existing antimicrobial compounds, only f luoroquinolones have a bactericidal effect against mycoplasmas, so use of these antimicrobials is preferable for the treatment of immunosuppressed patients. The limitation of the f luoroquinolones is the resistance of the causative agent to these antimicrobials. Mutations leading to amino acid substitutions in the composition of the targets of f luoroquinolones, gyrase and topoisomerase IV are the most common cause of resistance to f luoroquinolones both in mycoplasms and in other bacteria. It was noted that for mycoplasmas belonging to different species have different patterns of substitutions in the subunits of gyrase and topoisomerase IV. The differences of the structure of these proteins, ref lected in the natural susceptibility to f luoroquinolones in mycoplasmas, may be a reason of this heterogeneity. A number of studies indicate the existence of additional resistance mechanisms, which, first of all, include multiple-resistance systems. Such systems belonging to the ABC-transporter group were also found in mycoplasmas. They are described in Mycoplasma hominis and M. pneumoniae, in M. hominis, their ability to excrete f luoroquinolones from the cells was observed, and in M. pneumoniae the ability of multiple-resistance systems to export macrolides also was noted. Genes encoding components of multiple resistance systems have been found in genomes of other species, including M. genitalium and mycoplasmas, causing animal diseases. Also, in the non-pathogenic for human mycoplasmas Acholeplasma laidlawii, the ability to export proteins and genetic material associated with resistance to f luoroquinolones was found. Understanding the role of the mutations, the activity of transports and their cumulative effect in the development of resistance to f luoroquinolones is particularly important in the context of the determination of resistance to f luoroquinolones in difficult to culture pathogens M. genitalium and M. pneumoniae. Molecular methods for determining the resistance to antimicrobials are now included in clinical practice, but the lack of information about the molecular bases of mycoplasma resistance makes the result insufficiently informative for the pathogens of this group. In the review, the features of the development of resistance to f luoroquinolones in various species of human pathogenic mycoplasmas and their mechanisms at molecular level will be described.

Dual Targeting of Epidermal Growth Factor Receptor and HER3 by MEHD7945A as Monotherapy or in Combination with Cisplatin Partially Overcomes Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma Cell Lines

Aberrant signaling of the epidermal growth factor receptor (EGFR) plays a crucial role in the tumorigenesis of many cancer types, including head and neck squamous cell carcinoma (HNSCC), making it a compelling drug target. After initial promising results of EGFR-targeted therapies such as cetuximab, the problem of therapeutic resistance is emerging and new treatment options are necessary. In contrast to first-generation EGFR inhibitors, MEHD7945A (duligotuzumab) is a monoclonal antibody with dual EGFR/HER3 specificity. Consequently, treatment with MEHD7945A may result in a more pronounced therapeutic benefit. In this study, sensitivity to MEHD7945A as a single agent and in combination with cisplatin was investigated in cetuximab-sensitive and -resistant HNSCC cell lines under normal and reduced oxygen conditions. The results demonstrated that sensitivity to MEHD7945A was cell line dependent and influenced by oxygen concentration. An additive, but not synergistic, interaction between MEHD7945A and cisplatin was observed. In conclusion, MEHD7945A has the potential to partially overcome cetuximab resistance. Nevertheless, further research is warranted to determine additional resistance mechanisms to cetuximab treatment besides HER3 signaling. Unraveling these mechanisms will ultimately lead to the development of new therapeutic strategies to improve the response to EGFR blockage.

Characterization of a Sodium Channel Mutation in Permethrin-Resistant Rhipicephalus sanguineus (Acari: Ixodidae).

The brown dog tick, Rhipicephalus sanguineus (Latrielle) sensu lato, is an important ectoparasite of dogs and occasionally humans, capable of transmitting several pathogens, such as Rickettsia and Ehrlichia, which are of veterinary and medical importance. The brown dog tick is distributed worldwide and has an affinity for human habitations in much of its range. In some populations, lack of integrated pest management plans and overuse of pyrethroid pesticides and other sodium channel inhibitors has resulted in high levels of resistance to permethrin. Recently, a highly conserved region of the R. sanguineus sodium channel was sequenced, indicating that a single nucleotide polymorphism of thymine to cytosine on domain III segment VI of the sodium channel could confer resistance. A molecular assay targeting a point mutation in the sodium channel was developed and optimized to separate ticks expressing permethrin resistance from those from a susceptible colony. Thereafter, multiple field-collected phenotypically permethrin-resistant populations were evaluated using this molecular assay to determine genotype. As confirmed by DNA sequencing, a point mutation was present at a high rate in phenotypically resistant tick populations that was not present in the susceptible strain. These data suggest an additional permethrin resistance mechanism to metabolic resistance, which has been reported for this tick species, and confirm its association with phenotypic resistance. The results of this study further emphasize the need to preserve acaricide chemistry through rotation of active ingredients used to control ectoparasites.

Abstract 3198: Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer

Abstract Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades with few novel treatment options. We anticipate great potential for personalized targeted therapies, facilitated by recent characterizations of the mutational landscape of OSCCs. Combinations of targeted therapies may have greater efficacy through inhibiting compensatory pathways. For example, targeting EGFR has had limited success. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome-scale screening for drivers of sensitivity to EGFR-targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR-targeted therapy in some tumors. In complement, we again used CRISPR-Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR-targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway is a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, Chad Brenner. Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer [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 3198. doi:10.1158/1538-7445.AM2017-3198

Abstract 4092: Targeting compensatory mechanisms of resistance to phosphatidylinositol 3-kinase inhibitors in head and neck squamous cell carcinoma

Abstract Recent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite the frequency of activating mutations or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K), targeted inhibitors of PI3K have not shown clinical efficacy as monotherapies. We tested a panel of more than 20 patient-derived oral cavity squamous cell carcinoma (OCSCC) cell lines, which were profiled by Nimblegen V3 exome sequencing, and observed resistance to PI3K inhibitors despite PIK3CA copy number amplification and/or mutation. Of six inhibitors tested, only alpha-isoform selective and pan-PI3K agents were somewhat effective; these inhibitors, despite on-target and downstream activity, did not cause an appreciable reduction in cell viability when administered at submicromolar concentrations. We hypothesized that other oncogenic pathways might still be functional in the presence of PI3K inhibitors and might serve as mediators of this resistance. For example, our group and others have evaluated co-dependence on the Ras-MEK-ERK pathway in OCSCC, showing that this serves as a compensatory mechanism in some models. In order to more fully characterize other novel mechanisms of resistance, we have also developed a high-throughput screening approach that utilizes a resazurin cell viability assay. This screen serves as an unbiased means of testing ~1400 inhibitors as monotherapies and in combination with alpha-isoform selective PI3K inhibitor HS-173 and pan-PI3K inhibitor BKM120 in several OCSCC models. Further validation of “hits” from this screen, which are drugs that are effective in combination but not as monotherapies, may identify additional resistance mechanisms and lead to combination therapies to improve HNSCC patient prognosis. Citation Format: Nicole L. Michmerhuizen, Elizabeth Leonard, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Hui Jiang, Chad Brenner. Targeting compensatory mechanisms of resistance to phosphatidylinositol 3-kinase inhibitors in head and neck squamous 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 4092. doi:10.1158/1538-7445.AM2017-4092

O-001 - Systematic liquid biopsy identifies novel and heterogeneous mechanisms of acquired resistance in gastrointestinal (GI) cancer patients

Introduction: Understanding mechanisms of acquired resistance to targeted therapy can guide strategies to improve clinical outcome. Circulating tumor DNA (ctDNA) provides a non-invasive means to identify concurrent heterogeneous resistance mechanisms emerging during therapy. Methods: Since January 2015, 35 patients (pts) with molecularly-defined GI cancers (24 colorectal (CRC), 8 biliary, 3 gastroesophageal (GE)) achieving response or prolonged stable disease on targeted therapies had plasma collected at disease progression for next-generation sequencing of ctDNA. Molecular alterations identified were compared to ctDNA and/or tissue obtained pre-treatment to identify mechanisms of acquired resistance. When possible, post-progression tumor biopsies were also analyzed. Serial ctDNA specimens were evaluated to determine if the change in ctDNA levels can predict response to targeted therapy. Results: In 35 pts, at least one molecular mechanism of resistance was identified in progression ctDNA in 28 pts (80%) with 15 (43%) exhibiting >1 resistance alteration (range 2-12, median 3). Overall, 72 total resistance alterations were identified. 14 pts had matched progression tumor biopsies, and resistance alterations were identified in 9 (61%), all of which were detected in matched ctDNA. In 64% of these pts, additional resistance mechanisms not detected in the matched tumor biopsy were identified in ctDNA. In 7 pts with multiple progression tumor biopsies available, distinct metastases showed different resistance alterations, all of which were detectable in ctDNA, but in 6 (86%) of these pts, ctDNA detected additional resistance alterations not found despite multiple tumor biopsies, reflecting extensive heterogeneity. Several critical and/or novel resistance alterations were identified across tumor types treated with diverse therapies. In 14 of 17 RAS wildtype CRC pts receiving EGFR antibodies, 28 total (13 distinct) resistance alterations affecting KRAS, EGFR extracellular domain, MEK1, MET, and ERBB2 were detected. In 3 of 5 BRAF mutant CRC pts receiving BRAF inhibitor combinations, 10 total (9 distinct) resistance alterations affecting MAPK genes (KRAS, NRAS, BRAF, MEK1, MEK2) were detected. In a MET-amplified GE pt receiving a MET inhibitor, 2 novel secondary mutations in the MET kinase domain were identified. In 5 FGFR2 fusion-positive biliary pts receiving an FGFR inhibitor, all patients developed novel secondary FGFR2 kinase mutations (13 total, 8 distinct), with 2 pts harboring 5 concurrent mutations, and one harboring 2. In serial ctDNA specimens, the decrease in ctDNA levels by 4 weeks correlated with response and therapeutic outcome. Conclusion: Systematic ctDNA analysis at disease progression can effectively identify novel and heterogeneous mechanisms of acquired resistance in pts receiving targeted therapies for a range of molecularly-defined GI cancers. Compared to parallel tumor biopsies, ctDNA more effectively captured the heterogeneity of acquired resistance mechanisms, which may be important to guide subsequent therapy. Real-time monitoring of ctDNA levels may also represent a potential approach to predict response and resistance to therapy. Further studies to optimize integration of ctDNA in clinical decision-making are warranted.

Spectral analysis of nonequilibrium molecular dynamics: Spectral phonon temperature and local nonequilibrium in thin films and across interfaces

Although extensive experimental and theoretical works have been conducted to understand the ballistic and diffusive phonon transport in nanomaterials recently, direct observation of temperature and thermal nonequilibrium of different phonon modes has not been realized. Herein, we have developed a method within the framework of molecular dynamics to calculate the temperatures of phonon in both real and phase spaces. Taking silicon thin film and graphene as examples, we directly obtained the spectral phonon temperature (SPT) and observed the local thermal nonequilibrium between the ballistic and diffusive phonons. Such nonequilibrium also generally exists across interfaces and is surprisingly large, and it provides an additional thermal interfacial resistance mechanism. Our SPT results directly show that the vertical thermal transport across the dimensionally mismatched graphene/substrate interface is through the coupling between flexural acoustic phonons of graphene and the longitudinal phonons in the substrate with mode conversion. In the dimensionally matched interfaces, e.g. graphene/graphene junction and graphene/boron nitride planar interfaces, strong coupling occurs between the acoustic phonon modes on both sides, and the coupling decreases with interfacial mixing. The SPT method together with the spectral heat flux can eliminate the size effect of the thermal conductivity prediction induced from ballistic transport. Our work shows that in thin films and across interfaces, phonons are in local thermal nonequilibrium.

Knockdown resistance mutations predict DDT resistance and pyrethroid tolerance in the visceral leishmaniasis vector Phlebotomus argentipes.

BackgroundIndoor residual spraying (IRS) with DDT has been the primary strategy for control of the visceral leishmaniasis (VL) vector Phlebotomus argentipes in India but efficacy may be compromised by resistance. Synthetic pyrethroids are now being introduced for IRS, but with a shared target site, the para voltage-gated sodium channel (VGSC), mutations affecting both insecticide classes could provide cross-resistance and represent a threat to sustainable IRS-based disease control.Methodology/Principal findingsA region of the Vgsc gene was sequenced in P. argentipes from the VL hotspot of Bihar, India. Two knockdown resistance (kdr) mutations were detected at codon 1014 (L1014F and L1014S), each common in mosquitoes, but previously unknown in phlebotomines. Both kdr mutations appear largely recessive, but as homozygotes (especially 1014F/F) or as 1014F/S heterozygotes exert a strong effect on DDT resistance, and significantly predict survivorship to class II pyrethroids in short-duration bioassays. The mutations are present at high frequency in wild P. argentipes populations from Bihar, with 1014F significantly more common in higher VL areas.Conclusions/SignificanceThe Vgsc mutations detected appear to be a primary mechanism underlying DDT resistance in P. argentipes and a contributory factor in reduced pyrethroid susceptibility, suggesting a potential impact if P. argentipes are subjected to suboptimal levels of pyrethroid exposure, or additional resistance mechanisms evolve. The assays to detect kdr frequency changes provide a sensitive, high-throughput monitoring tool to detecting spatial and temporal variation in resistance in P. argentipes.

Plasmid-mediated quinolone resistance determinants among Gram-negative bacteraemia isolates: a hidden threat.

The aim of the study was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in an unselected collection of bloodstream isolates recovered over an 18-month period in a laboratory affiliated to a university hospital in Athens, Greece, and to assess their impact on the in vitro activity of ciprofloxacin and levofloxacin. Eight PMQR genes were screened by PCR and sequencing. All PMQR-positive isolates were submitted to isoelectric focusing for β-lactamase detection, conjugation or transformation, time-kill assays, mutant prevention concentrationand inoculum effect evaluation. PCR and sequencing of gyrA and parC were performed for detection of chromosomal mutations. Among 96 Gram-negative isolates, 7 (7.3 %) carried one or more PMQR genes. qnrS1 was the most prevalent (5.2 %), followed by aac(6')-Ib-cr (4.2 %) and their combination (2 %). Cloning was successful for three isolates. The presence of a single PMQR determinant without any target modification was not associated with quinolone resistance with one exception, Stenotrophomonasmaltophilia carrying qnrS1, which was resistant to norfloxacin and ciprofloxacin, but in this isolate, additional mechanisms of quinolone resistance cannot be excluded. All PMQR-positive isolates showed a significant inoculum effect. The mutant prevention concentrations of ciprofloxacin against the quinolone-susceptible clinical isolates ranged from 0.38 to 32 mg l-1 and those of levofloxacin from 1 to 32 mg l-1. PMQRs compromised the bactericidal activity of ciprofloxacin and levofloxacin when expressed in Enterobactercloacae, S. maltophilia or Klebsiellapneumoniae and when more than one co-existed. PMQR determinants represent an unrecognized threat, capable to compromise the in vitro activity of quinolones if expressed in a favourable genetic environment and to favour selection of resistant mutants by widening the mutant selection window of these agents.

Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis.

ABSTRACTPreviously, we showed that mutations in Mycobacterium tuberculosis panD, involved in coenzyme A biosynthesis, cause resistance against pyrazinoic acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against pyrazinoic acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.

FGF2 from Bone Marrow Stroma Protects Acute Myeloid Leukemia Cells from the FLT3 Inhibitor Quizartinib and Facilitates Acquisition of Resistance Mutations

Background: Acute myeloid leukemia (AML) patients with internal tandem duplication of FLT3 (FLT3-ITD AML) routinely develop resistance to FLT3 inhibitor monotherapy. Resistance to inhibitors such as quizartinib can develop through mutation of the FLT3 kinase domain, but not all resistant patients have mutations indicating that additional mechanisms of resistance are important. Although quizartinib induces a rapid reduction of circulating leukemia blasts, residual blasts in the marrow persist, suggesting that the microenvironment protects blasts from quizartinib and may serve as a reservoir for development of resistance.Results: Using a screen of molecules from the microenvironment, we found that FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) were the two most protective proteins of FLT3-ITD MOLM14 cells when treated with 10 nM quizartinib in vitro. FL protection has been previously described by Mark Levis' group, so we focused on the mechanism of FGF2 protection. FGF2 bound FGFR1, activating the MAPK pathway to mediate resistance. FGF2-mediated resistance could be overcome by concomitant inhibition of the FGFR receptor using the inhibitor PD173074 or by siRNA knock-down of FGFR1. FGFR1 is highly expressed in primary AML cells, and exogenous FGF2 protected primary FLT3-ITD AML cells from quizartinib ex vivo in a dose-dependent fashion (p=0.0007 at 100 ng/ml).MOLM-14 cells were cultured continuously with quizartinib in media alone, or media supplemented with 10 ng/ml FGF2 or FL to mimic the effects of the microenvironment. MOLM14 cells supplemented with FL or FGF2 resumed growth after 6-8 weeks, significantly faster than cells cultured in media alone, of which only 2/4 developed resistance after 12 weeks. To test the dependence of resistance cultures on exogenous ligand, we removed ligand from the FGF2- and FL-dependent cultures after 4 months and continued quizartinib treatment. After a temporary pause in growth, the cells regained exponential growth within 1 month. We assayed FLT3 resistance mutations by Sanger sequencing during this time, and again at 8 months with a targeted next-generation sequencing panel of commonly mutated genes in AML. Deep sequencing revealed multiple FLT3 mutations along with recurrent mutations of KRAS and NRAS, indicating the importance of the FLT3 and MAPK signaling pathway in resistance (Figure 1). Multiple FLT3 mutations were identified strongly suggesting that FLT3 mutations were not pre-existing but developed during culture with quizartinib (Figure 1). In contrast, the frequency of the KRAS G13D mutation suggested this mutation was present at a low frequency in MOLM14 cells.FGF2 expression was evaluated by immunohistochemistry in serial bone marrow core biopsies from 10 FLT3-ITD AML patients on the phase II quizartinib trial. Stromal FGF2 expression increased significantly with quizartinib therapy (p<0.01) and remained elevated until patients acquired FLT3 resistance mutations or other intrinsic mechanisms of resistance. Once stromal FGF2 became redundant for survival, expression decreased again. FGF2 protected cells in a paracrine fashion since FGF2 immunofluorescence did not overlap with CD45 (hematopoietic marker) in core biopsies of quizartinib-treated patients.To test the effect of combined FLT3 and FGFR inhibition in a more complex in vitro model of the microenvironment, MOLM14 cells were co-cultured in transwells over HS-5 (FGF2 high expression) or HS-27 stromal cell lines (FGF2 low). HS-5 stromal cells were highly protective of MOLM14 cells treated with quizartinib alone, but this protection was attenuated by combined FGFR and FLT3 inhibition (p<0.01). In contrast, protection of MOLM14 cells in HS-27 co-culture was minimal, and unaffected by combined FLT3 and FGFR inhibition.Conclusions: Our data supports a two-step model of resistance to quizartinib with initial resistance mediated by microenvironmental proteins such as FGF2 and/or FL, followed by kinase domain mutation of FLT3 and/or activating mutations of the RAS pathway. Early resistance mediated by FGF2-expressing stroma can be overcome by concomitant inhibition of FLT3 and FGFR suggesting that pre-emptively targeting extrinsic resistance pathways, in combination with newer FLT3 inhibitors that have activity against kinase domain mutations, will improve the durability of response to FLT3 inhibitors. [Display omitted] DisclosuresAgarwal:CTI BioPharma Corp: Research Funding. Kovacsovics:Seattle Genetics: Research Funding. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Resistant T-Cell Acute Lymphoblastic Leukemias That Emerge after In Vivo Treatment with Dexamethasone Frequently Down-Regulate Glucocorticoid Receptor Protein Expression

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, with T lineage ALL (T-ALL) accounting for approximately 20% of cases. Many T-ALL patients present with high risk clinical features, and receive aggressive multi-agent genotoxic therapies that pose a substantial risk of long-term adverse health effects. Glucocorticoids (GCs) are an integral part of current treatment strategies for T-ALL and other lymphoid cancers. Despite decades of clinical use, the molecular mechanisms underlying GC efficacy and resistance are incompletely understood. Limited initial response to GCs correlates with poor outcome, and secondary GC resistance is frequently observed in relapsed patients. Thus, identifying drugs that either enhance initial responses or prevent resistance to GCs could significantly improve the treatment of T-ALL and other lymphoid malignancies. Our laboratory performed retroviral insertional mutagenesis (RIM) in wild-type (KrasWT) and KrasG12D "knock-in" mutant mice to generate a panel of primary transplantable T-ALLs that recapitulate the genetic heterogeneity found in human cancers. We used these reagents to assess the efficacy of treatment with the GC dexamethasone (DEX) alone and in combination with the PI3 kinase (PI3K) inhibitor GDC-0941. We observed a robust and significant overall response to 15 mg/kg/day DEX in cohorts of recipient mice transplanted with 10 independent parental KrasWT (n=5) and KrasG12D (n=5) T-ALLs, which was modestly enhanced by combined treatment with 125 mg/kg/day GDC-0941. Prolonged in vivo treatment resulted in outgrowth of 65 independent relapsed T-ALLs, many of which harbor novel retroviral integrations. We verified intrinsic drug resistance in a number of these relapsed leukemias by transplanting them into secondary recipients and treating these mice with 15 mg/kg/day DEX. Intriguingly, 23 (35%) of the T-ALLs that relapsed after an initial response to treatment exhibited markedly reduced GC receptor (GR) protein expression, suggesting a novel and common mechanism for evading GC-induced cell death. Similarly, analysis of T-ALL patient samples showed that low GR expression is rare in diagnostic specimens, but enriched in relapsed/refractory cases. We have identified two likely mechanisms of GR down-regulation in relapsed mouse T-ALLs including a nonsense mutation in the gene encoding GR and a retroviral integration in a putative distal GR promoter region that greatly reduces mRNA expression. We also performed transcriptome (RNA-seq) analysis in one sensitive parental and corresponding resistant KrasWT T-ALL after short-term in vivo DEX treatment, and observed a dramatic reduction in the number of differentially expressed genes in the resistant versus parental leukemia. We recently generated transcriptomes from this panel of 10 primary RIM-induced T-ALLs with the goal of identifying a GC response signature, and also to assess the ability of resistant cells that have lost or retained GR expression to activate this program. Finally, in contrast to previous studies in this genetically accurate in vivo preclinical model (Dail et al. Nature 2014), very few relapsed T-ALLs showed reduced Notch intracellular domain (NICD) expression relative to the corresponding parental leukemia suggesting that loss of Notch1 activation is not a major mechanism contributing to DEX resistance. Taken together, these data reveal an unexpected and common putative mechanism of GC resistance in T-ALL that can inform the development of treatment strategies for relapsed/refractory patients. We are currently focusing on analyzing transcriptome data and performing whole exome sequencing in order to uncover additional mechanisms of resistance to DEX, and then using these data to identify and investigate targeted inhibitors that might overcome GC resistance in vivo. DisclosuresDail:Genentech, Inc.: Employment. Sampath:Genentech: Employment.

Upregulation of MAPK/MCL-1 Maintaining Mitochondrial Oxidative Phosphorylation Confers Acquired Resistance to BCL-2 Inhibitor Venetoclax in AML

ABT-199 (venetoclax), a selective small-molecule antagonist of the anti-apoptotic protein BCL-2, enables the activation of pro-apoptotic proteins and the induction of cancer cell death. Our previous studies found that AML is a BCL-2 dependent disease and responds robustly to venetoclax by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). Despite initial responses to single agent venetoclax in a Phase II trial of relapsed AML, patients ultimately developed resistance and progressed (Konopleva et al., Cancer Discovery 2016). In this study we investigated mechanisms of acquired resistance to venetoclax in preclinical AML models.First, we generated 5 AML cell lines resistant to ³1µM venetoclax. No BAX (exon5 and 6) or BCL2 (exon2) mutations were found in resistant cells. Immunoblotting analysis demonstrated increased expression of anti-apoptotic proteins MCL-1, BCL-2 A1, and BCL-XL, and a decrease of pro-apoptotic PUMA protein in selected resistant cell lines. To probe the functional interactions between the pro- and anti-apoptotic proteins, we next performed co-immunoprecipitation (co-IP) studies. The anti-BIM and anti-MCL-1 co-IPs revealed reduced levels of BIM:BCL-2 complexes and increased BIM:MCL-1 complexes in resistant cells compared to their parental counterparts (Fig 1B). The BH3 profiling technique examines mitochondrial sensitivity to different BH3 mimetic peptides, and has proven to be a useful tool to determine cell dependence on anti-apoptotic BCL-2 family proteins. BH3 profiling demonstrated that resistant cells had increased responses to NOXA, MS1 and HRK peptides, indicating increased dependence on MCL-1 and/or BCL-XL (Fig 1C). To characterize the functional role of MCL-1 in resistance to venetoclax, we co-treated parental and resistant cells with selective BCL-XL or MCL-1 inhibitors A-1155463 (Leverson et al. Science Transl Med 2015) and A-1210477 (Leverson et al., Cell Death Dis 2015). The combination of venetoclax with either A-1155463 or A-1210477 showed synergistic growth inhibition in all 5 parental cell lines. Notably, 4 of the 5 resistant cell lines (OCI-AML2, Kasumi, MV4-11, MOLM13) became more sensitive to an MCL-1 inhibitor but not to a BCL-XL inhibitor (Fig 1E). However, no further sensitization was seen in combination with venetoclax in resistant cells.To characterize additional mechanisms of resistance to venetoclax in AML cells, we conducted RNA sequencing of single cell clones (2 clones/cell line) isolated from paired isogenic cells (OCI-AML2, MV4-11, MOLM13). Analysis of RNA expression patterns by gene set enrichment analysis (GSEA) revealed elevated expression of genes in the RAS/MAPK pathway (Fig 1F), consistent with increased p-ERK and p-p90-RSK protein levels (Fig 1G). Inhibition of MAPK with MEK inhibitor GDC-0973 reduced MCL-1 expression in parental but not in resistant cells, indicating that MAPK activation partially contributed to high MCL-1 levels (Fig 1G).GSEA of RNAseq data further uncovered altered expression of genes involved in mitochondrial oxidative phosphorylation (OxPhos) in 3 resistant cell lines with high MCL-1 expression (OCI-AML2, MV4-11 and MOLM-13). Notably, BCL-2 was reported to sustain AML stem cell survival through maintenance of the mitochondrial activity of OxPhos (Lagadinou etal., Cell Stem Cell, 2013). Analysis of mitochondrial respiration using a Seahorse Bioanalyzer demonstrated similar levels of oxygen consumption rate (OCR) in parental and resistant cells. Inhibition of BCL-2 with 100nM venetoclax for only 2 hrs. fully blocked baseline and maximal respiratory activity in parental but not in resistant cells. In turn, inhibition of MCL-1 with A-1210477 inhibited respiration in both parental and resistant cells, indicating a role for MCL-1 in sustaining mitochondrial activity in venetoclax-resistant AML cells, which can maintain unperturbed mitochondrial function.In summary, we identified a novel mechanism of resistance to targeted BCL-2 inhibition through upregulation of MAPK leading to increased levels of anti-apoptotic MCL-1 that binds and neutralizes BIM and maintains the mitochondrial OxPhos pathway in AML cells. Concomitant inhibition of BCL-2 and MCL-1, or of BCL-2 and OxPhos could induce synergistic cell death in AML and conceivably prevent the emergence of venetoclax resistance. [Display omitted] DisclosuresTyner:Constellation Pharmaceuticals: Research Funding; Janssen Research & Development: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; Inctye: Research Funding; Seattle Genetics: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Phosphoproteomics Reveals MAPK Inhibitors Enhance MET- and EGFR-Driven AKT Signaling in KRAS-Mutant Lung Cancer.

This study highlights the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K/AKT signaling when the MAPK pathway is inhibited. Mol Cancer Res; 14(10); 1019-29. ©2016 AACR.

Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors.

Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance.

Genetic environment of the KPC gene in Acinetobacter baumannii ST2 clone from Puerto Rico and genomic insights into its drug resistance.

Carbapenems are considered the last-resort antibiotics to treat infections caused by multidrug-resistant Gram-negative bacilli. The Klebsiella pneumoniae carbapenemase (KPC) enzyme hydrolyses β-lactam antibiotics including the carbapenems. KPC has been detected worldwide in Enterobacteriaceae and Pseudomonas aeruginosa isolates associated with transposon Tn4401 commonly located in plasmids. Acinetobacter baumannii has become an important multidrug-resistant nosocomial pathogen. KPC-producing A. baumannii has been reported to date only in Puerto Rico. The objective of this study was to determine the whole genomic sequence of a KPC-producing A. baumannii in order to (i) define its allelic diversity, (ii) identify the location and genetic environment of the blaKPC and (iii) detect additional mechanisms of antimicrobial resistance. Next-generation sequencing, Southern blot, PFGE, multilocus sequence typing and bioinformatics analysis were performed. The organism was assigned to the international ST2 clone. The blaKPC-2 was identified on a novel truncated version of Tn4401e (tentatively named Tn4401h), located in the chromosome within an IncA/C plasmid fragment derived from an Enterobacteriaceae, probably owing to insertion sequence IS26. A chromosomally located truncated Tn1 transposon harbouring a blaTEM-1 was found in a novel genetic environment within an antimicrobial resistance cluster. Additional resistance mechanisms included efflux pumps, non-β-lactam antibiotic inactivating enzymes within and outside a resistance island, two class 1 integrons, In439 and the novel In1252, as well as mutations in the topoisomerase and DNA gyrase genes which confer resistance to quinolones. The presence of the blaKPC in an already globally disseminated A. baumannii ST2 presents a serious threat of further dissemination.

Abstract 2127: Identification of a novel NAMPT inhibition resistance mechanism utilizing the de novo NAD+ biosynthesis pathway

Abstract Targeting NAD(H) metabolism is being interrogated as a novel anti-cancer strategy. Nicotinamide phoshophoribosyltransferase (NAMPT) was found to be overexpressed in tumor cells, and plays a key role in the replenishment of the NAD(H) pool in cells. In this study we have used a commercially available NAMPT inhibitor, GMX1778, which exerts a cytotoxic effect by decreasing the cellular level of NAD(H). Through serial selection with increasing sub-lethal concentrations of GMX1778, we derived a GMX1778-resistant HT1080 human fibrosarcoma cell line (HT1080-GMX). The IC50 value for GMX1778-induced cell killing of the resistant cell line was 100-fold greater than that determined for the parental cell line. No significant change in the level of expression of NAMPT was observed in HT1080 vs. HT1080-GMX cells. However, a key enzyme in the de novo NAD(H) synthesis pathway known as quinolinate phosphoribosyl transferase (QPRT) was significantly upregulated as validated by Western blot and bDNA analysis. Knockdown of QPRT partially restored the sensitivity to GMX1778 exposure in HT1080-GMX cells, but did not affect the parental cells. The cytotoxicity of GMX1778 can be partially rescued with exogenous quinolinic acid, which permits NAD(H) repletion via QPRT in the de novo pathway, in HT1080-GMX cells but not HT1080 parental cells. Overexpression of QPRT in HT1080 cells did not fully rescue GMX-induced cytotoxicity and strongly suggests that there are additional mechanisms of resistance in the HT1080-GMX cell line beyond QPRT overexpression. Exome sequencing of the NAMPT gene in the HT1080-GMX resistant lines identified a single mutation of amino acid residue 18 from tyrosine to cysteine [NAMPT(Y18C)] located in the first exon of one allele. Tyrosine 18 is located near the active site of NAMPT and associates with NAMPT small molecule inhibitors through a pi stacking interaction, as determined by studies of the enzyme's crystal structure. Overexpression of the NAMPT mutant Y18C in HT1080 cells significantly reduced the sensitivity to GMX1778 exposure, but overexpression of a NAMPT Y18F mutant had no effects compared to the parental line. Based on our results, activation of an alternate NAD(H) biosynthesis pathway via QPRT or the Y18C mutation within the NAMPT coding sequence can both be contributors to resistance in this line. The characterization of resistance inducing up-regulation of other sources of NAD(H) such as de novo synthesis or mutations in NAMPT may be useful in developing next-generation NAMPT inhibitors that are less affected by known resistance mechanisms. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: jun guo, Chris Tse, William N. Pappano. Identification of a novel NAMPT inhibition resistance mechanism utilizing the de novo NAD+ biosynthesis pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2127.

Abstract 880: Mapping the metastatic colorectal cancer phospho-proteome for predicting response to cetuximab

Abstract Introduction: The discovery of the key role of the epidermal growth factor receptor (EGFR) and its downstream signaling effectors in the pathophysiology of colorectal cancer (CRC) has resulted in the clinical use of targeted therapies in the treatment of metastatic CRC (mCRC). However, clinical benefit to EGFR blockade is observed in only a subgroup of CRC patients wild type for KRAS andNRAS. Genomic characterization of patient derived xenograft (PDX) models of mCRC have revealed additional resistance mechanisms to the EGFR MAb cetuximab including amplification of MET, HER2 and mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 (Bertotti et al. 2011, 2015). However, not all resistance is understood and the biochemical signaling correlates of sensitivity and resistance remain incompletely understood. Phosphoproteomics allows for comprehensive protein phosphorylation profiling, and thereby may shed new insights into the biology of CRC and the molecular basis of cetuximab response. Aim: To identify aberrant signaling pathways, predictive biomarkers, and alternative drug targets in genomically-annotated CRC PDX models by mass spectrometry-based phosphoproteomics. Methods: We performed LC-MS/MS based phosphoproteome profiling (van der Mijn et al. 2015; Piersma et al. 2015) on untreated CRC PDX models lacking KRAS, NRAS or BRAF mutation (n = 20). For comparison, KRAS, BRAF or NRAS-mutant and MET or HER2 amplified models (n = 21) were analyzed. Phosphopeptides were captured by phosphotyrosine antibodies and titanium dioxide. MS/MS data was searched using MaxQuant. Phosphoproteome data (ion intensities and spectral counts) were correlated with drug sensitivity. Dedicated bioinformatics was used to identify hyperactive kinases and signaling pathways in individual tumors. Results: The dataset contained 13.418 tyrosine-, serine- and threonine-phosphorylated peptides derived from 4588 proteins, including 237 phosphorylated kinases. Unsupervised hierarchical clustering of phosphopeptide intensities revealed subclusters enriched for sensitive and resistant models. Among cetuximab-resistant models, HER2 and MET were identified as hyperphosphorylated and hyperactive driver kinases, confirming the genomic annotation. Sensitivity to combination treatment of both EGFR and driver kinases was observed previously, providing proof-of-principle of the use of phosphoproteomics for the identification of driver kinases. Conclusion/Future: This project is uncovering the phosphoproteomic landscape of colorectal tumors and will yield a better understanding of signaling pathways involved, with an emphasis on identifying activated drug targets and predictive biomarkers in this disease, in relation to KRAS/NRAS/BRAF status and response to anti-EGFR therapy. Citation Format: Robin Beekhof, Mariette Labots, Jaco C. Knol, Tim R.A. Schelfhorst, Nicole van Grieken, Sander R. Piersma, Thang V. Pham, Andrea Bertotti, Livio Trusolino, Henk M.W. Verheul, Connie R. Jiménez. Mapping the metastatic colorectal cancer phospho-proteome for predicting response to cetuximab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 880.

P53 Loss in MYC-Driven Neuroblastoma Leads to Metabolic Adaptations Supporting Radioresistance.

Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. Mutations in the p53 (TP53) tumor suppressor are rare at diagnosis, but evidence suggests that p53 function is often impaired in relapsed, treatment-resistant disease. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ER(TAM) fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53(KI)). We observed no significant differences in tumor-free survival between Th-MYCN mice heterozygous for Trp53(KI) (n = 188) and Th-MYCN mice with wild-type p53 (n = 101). Conversely, the survival of Th-MYCN/Trp53(KI/KI) mice lacking functional p53 (n = 60) was greatly reduced. We found that Th-MYCN/Trp53(KI/KI) tumors were resistant to ionizing radiation (IR), as expected. However, restoration of functional p53ER(TAM) reinstated sensitivity to IR in only 50% of Th-MYCN/Trp53(KI/KI) tumors, indicating the acquisition of additional resistance mechanisms. Gene expression and metabolic analyses indicated that the principal acquired mechanism of resistance to IR in the absence of functional p53 was metabolic adaptation in response to chronic oxidative stress. Tumors exhibited increased antioxidant metabolites and upregulation of glutathione S-transferase pathway genes, including Gstp1 and Gstz1, which are associated with poor outcome in human neuroblastoma. Accordingly, glutathione depletion by buthionine sulfoximine together with restoration of p53 activity resensitized tumors to IR. Our findings highlight the complex pathways operating in relapsed neuroblastomas and the need for combination therapies that target the diverse resistance mechanisms at play. Cancer Res; 76(10); 3025-35. ©2016 AACR.