Innately Resistant Research Articles

Abstract B032: Deciphering the mechanisms of environmentally mediated Alectinib resistance in ALK+NSCLC

Abstract Non-small cell lung cancer (NSCLC) is a devastating disease with a subset containing targetable oncogenic mutations that strongly benefit from targeted therapies. 3-7% of NSCLC patients bear an amplification of the Anaplastic lymphoma kinase (ALK) and Echinoderm microtubule-associated protein like 4 (EML4) fusion gene. These tumors typically show strong durable responses to first line therapy, Alectinib, an ALK tyrosine kinase inhibitors (ALK-TKIs). However, there is a high degree of patient-to-patient variability in the level of response to this drug; with some patients being innately resistant or less sensitive. Moreover, weak initial sensitivity to therapy has been linked to poor long-term responses with early acquisition of resistance. Thus, to improve clinical outcomes, we need to understand the mechanisms that lead to this reduced sensitivity to ALK inhibition. While, historically, reduced sensitivity to ALK TKI was thought to reflect cell intrinsic mechanisms, a growing body of evidence points to the importance of tumor microenvironmentally (TME) mediated mechanisms. We fortuitously encountered an observation of strong environment-mediated desensitization to Alectinib. We found that combining Alectinib with a small molecule inhibitor, Jumonji histone demethylase inhibitor, JIB04 in vitro overcomes tolerance, preventing ALK+NSCLC cells from developing resistance to Alectinib. Surprisingly, our validation experiments in mouse models revealed the opposite effect, where, JIB04 desensitized tumors to ALK inhibition. The disparity in the in vitro versus the in vivo response indicates the contribution of the TME in the reduced Alectinib sensitivity observed in vivo. The resistance phenotype in Alectinib+JIB04 treated mice was strongly associated with extensive inflammation in the tumor proximal tissues, along with higher circulating levels of inflammatory markers, IL6, LIF and monocytes, suggesting that inflammation may play a role in mediating this resistance. This is consistent with the recent findings where the markers of systemic inflammation such as Neutrophil to Lymphocyte ratio (NLR) have been associated with poor prognosis in ALK+NSCLC. While the mechanistic understanding is not well characterized, our model may help us explore the mechanisms of this correlation. Thus, our objective is to understand the underlying microenvironmental mechanism of this resistance to Alectinib. We hypothesize that Alectinib+JIB04 associated inflammation reduces ALK+NSCLC’s sensitivity to Alectinib. We envision two possibilities, either inflammation may directly affect resistance to Alectinib or indirectly where inflammation enhances tumor cell plasticity that then leads to resistance. We posit that the underlying molecular mechanisms of Alectinib+JIB04 mediated resistance are likely to be relevant to a subset of patients with poor initial responses with weak sensitivity or innate resistance to Alectinib observed in the clinics and will improve our knowledge about microenvironmental regulation of resistance. Citation Format: Pragya Kumar, Virginia Turati, Andriy Marusyk. Deciphering the mechanisms of environmentally mediated Alectinib resistance in ALK+NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B032.

Mechanistic basis and efficacy of targeting the β-catenin–TCF7L2–JMJD6–c-Myc axis to overcome resistance to BET inhibitors

AbstractThe promising activity of BET protein inhibitors (BETi's) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin–TCF7L2–JMJD6–c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin–TCF7L2–JMJD6–c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

Overcoming Innate Resistance to a Beta-Catenin Inhibitor-Tegavivint- by Manipulating Autophagy in Multiple Myeloma

Multiple myeloma (MM) is an incurable haematological malignancy with patients eventually exhausting all available therapeutic modalities and succumbing to the disease. Various lines of evidence have demonstrated that the Wnt/β-catenin pathway is frequently up-regulated in advanced MM. Tegavivint (BC2059), an inhibitor of the Wnt/β-catenin pathway, that we have previously shown to have anti-MM activity in vitro, ex vivo and in vivo, is currently in clinical trials for the treatment of solid tumours. In this study, we further demonstrate the dose dependent pro-apoptotic activity of BC2059, including activity against the innately resistant human MM cell line (HMCL) LP1. We show that BC2059 causes down-regulation of β-catenin and it’s down-stream target survivin, in parallel with reactive oxygen species (ROS) generation. Coincident with this, BC2059 induces autophagy with a rise in LC3II and fall in p62. Moreover, chemical inhibition of autophagy was highly synergistic with BC2059 in all HMCL tested, with evidence of pro-apoptotic potentiation of BC2059 activity, with enhanced generation of active caspases 8, 9 and 3. Consistent with this, BC2059 treatment of LP1 in combination with knock down of the autophagy related protein Atg5 markedly enhanced cell killing. In conclusion, our results demonstrate that resistance to BC2059 can be partially attributed to the induction of cytoprotective autophagy and that inhibition of autophagy enhances BC2059-induced MM cell killing. The combination of Wnt/β-catenin pathway and autophagic inhibition represents a novel therapeutic strategy for advanced MM that warrants further evaluation.

Rethink of EGFR in Cancer With Its Kinase Independent Function on Board.

The epidermal growth factor receptor (EGFR) is one of most potent oncogenes that are commonly altered in cancers. As a receptor tyrosine kinase, EGFR's kinase activity has been serving as the primary target for developing cancer therapeutics, namely the EGFR inhibitors including small molecules targeting its ATP binding pocket and monoclonal antibodies targeting its ligand binding domains. EGFR inhibitors have produced impressive therapeutic benefits to responsive types of cancers. However, acquired and innate resistances have precluded current anti-EGFR agents from offering sustainable benefits to initially responsive cancers and benefits to EGFR-positive cancers that are innately resistant. Recent years have witnessed a realization that EGFR possesses kinase-independent (KID) pro-survival functions in cancer cells. This new knowledge has offered a different angle of understanding of EGFR in cancer and opened a new avenue of targeting EGFR for cancer therapy. There are already many excellent reviews on the role of EGFR with a focus on its kinase-dependent functions and mechanisms of resistance to EGFR targeted therapies. The present opinion aims to initiate a fresh discussion about the function of EGFR in cancer cells by laying out some unanswered questions pertaining to EGFR in cancer cells, by rethinking the unmet therapeutic challenges from a view of EGFR's KID function, and by proposing novel approaches to target the KID functions of EGFR for cancer treatment.

Overexpression of Insulin‐like Growth Factor Binding Protein 1 (IGFBP1) Generates Tamoxifen Resistance in Breast Cancer Cells

Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα‐positive breast cancer. Acquired resistance to tamoxifen remains a clinically relevant challenge for many patients receiving treatment and some patients with ERα‐positive breast cancer are innately resistant to tamoxifen treatment. The molecular mechanisms underlying both acquired and innate tamoxifen resistance are not well understood. Previous studies have shown that, in addition to ERα inhibition, tamoxifen activates the G‐protein‐coupled estrogen receptor 1 (GPER1) and activation of this receptor is associated with the development of tamoxifen resistance. GPER1 mediates the extracellular accumulation of the insulin‐like growth factor binding protein 1 (IGFBP‐1) after tamoxifen treatment resulting in the inhibition of the IGF‐1R signaling in breast cancer cells. We hypothesize sustained exposure of breast cancer cells to IGFBP‐1 will result in the development of tamoxifen resistance. Stable transfection of an IGFBP‐1 expression vector was used for sustained exposure of MCF‐7 breast cancer cells. IGF‐1R expression was decreased in MCF‐7 cells stably transfected with the IGFBP‐1 expression vector similar to IGF‐1R expression in tamoxifen resistant breast cancer cells. Similar to tamoxifen‐resistant MCF‐7 cells, the IGFBP‐1 overexpressing MCF‐7 cells also have elevated levels of activated ERK1/2. Finally, cell viability assays revealed that sustained IGFBP‐1 overexpression decreased breast cancer sensitivity to tamoxifen treatment. Taken together, these results suggest that elevated levels of IGFBP‐1 contribute to the development of tamoxifen resistance in breast cancer cells.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B108: The HDAC inhibitor HBI-8000 enhances immunotherapy with either PD-1 or PD-L1 blockade in the MC38 model of colon cancer

Abstract Immunomodulatory antibodies targeting the immune checkpoint receptors CTLA-4, PD-1 and its ligand PD-L1, have resulted in significantly improved clinical benefits over the standard of care in many cancer types. While these immune checkpoint inhibitors have successfully exploited the adaptive immune system to produce remarkable clinical responses in various cancers, a significant number of patient's tumors are either innately resistant or develop resistance, and the disease eventually progresses. For this reason, rational drug combinations with checkpoint inhibitors and other immunomodulating agents are imperative to improve the rate of durable responses and patient survival. Histone deacetylase inhibitors (HDACi) have been successful as monotherapy for hematologic malignancies, but their clinical effectiveness against solid tumors has been limited to date. The long-held belief that HDACi are immunosuppressive has been challenged recently by data demonstrating the ability of HDACi to enhance anti-tumor immunity through effects on tumor immunogenicity, regulatory T cells, myeloid-derived suppressor cells (MDSCs), as well as effector cell functions. HBI-8000 (chidamide) is a novel, orally bioavailable Class I (HDAC 1,2,3) and Class II (HDAC 10) selective HDACi with a demonstrated positive effects on antitumor immunity, enhancing the activity of CTL and NK cells, as well as having direct activity against tumor cells through reversal of apoptosis resistance. We report here that HBI-8000 enhances the activity of both PD-1 and PD-L1 blocking antibody (Ab) in the MC38 syngeneic mouse model of colon cancer. Combinations of HBI-8000 with PD-1 or PD-L1 Ab resulted in statistically significant decreases in tumor progression and increases in survival compared to single agent therapy. Analysis of tumor infiltrating lymphocytes (TIL) revealed a significant increase in the percentage of intratumoral CD8+ T cells in tumors treated with both HBI-8000/PD-1 Ab and HBI-8000/PD-L1 Ab combinations. The TIL analysis also revealed a trend towards lower percentages of Tregs and granulocytic MDSCs. To evaluate the potential role of this combination as a 2nd line treatment option, i.e., in subjects failing 1st line checkpoint antibody therapy, we developed a model of immune checkpoint antibody failure, where tumor-bearing mice were initially treated (1st line) with either PD-1 or PD-L1 blocking Ab. Mice which had either slow disease progression or stable disease followed by progression, were randomized into one of six 2nd line treatment groups, including combinations of anti PD-1 or PD-L1 blocking Ab with HBI-8000. Fifty percent of mice failing 1st line PD-1 Ab treatment experienced a complete response, and were rescued by treatment with HBI-8000 combined with PD-L1 Ab. Altogether our data suggest that the addition of the HDACi HBI-8000 can improve the anti-tumor response to either PD-1 or PD-L1 blocking antibodies, increase the percentage of intratumoral immune effector cells and potentially counter innate resistance to checkpoint inhibitors. Our data also suggests that combination therapy with HBI-8000 and a checkpoint inhibitor can rescue tumor-bearing mice that have failed 1st line therapy with different checkpoint inhibitor. These results provide a strong rationale for the evaluation of therapeutic regimens utilizing PD-L1 or PD-1 blocking antibodies in combination with HBI-8000. Citation Format: Reid P. Bissonnette, Alain Rolland, Bob Goodeneow, Mireille Gillings. The HDAC inhibitor HBI-8000 enhances immunotherapy with either PD-1 or PD-L1 blockade in the MC38 model of colon cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B108.

Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma

PD-1 immune checkpoint blockade provides significant clinical benefits for melanoma patients. We analyzed the somatic mutanomes and transcriptomes of pretreatment melanoma biopsies to identify factors that may influence innate sensitivity or resistance to anti-PD-1 therapy. We find that overall high mutational loads associate with improved survival, and tumors from responding patients are enriched for mutations in the DNA repair gene BRCA2. Innately resistant tumors display a transcriptional signature (referred to as the IPRES, or innate anti-PD-1 resistance), indicating concurrent up-expression of genes involved in the regulation of mesenchymal transition, cell adhesion, extracellular matrix remodeling, angiogenesis, and wound healing. Notably, mitogen-activated protein kinase (MAPK)-targeted therapy (MAPK inhibitor) induces similar signatures in melanoma, suggesting that a non-genomic form of MAPK inhibitor resistance mediates cross-resistance to anti-PD-1 therapy. Validation of the IPRES in other independent tumor cohorts defines a transcriptomic subset across distinct types of advanced cancer. These findings suggest that attenuating the biological processes that underlie IPRES may improve anti-PD-1 response in melanoma and other cancer types.

MS4A3 Improves Imatinib Response and Survival in BCR-ABL1 Primary TKI Resistance and in Blastic Transformation of Chronic Myeloid Leukemia

Background: Mutations in the BCR-ABL1 kinase domain are a well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs), but less is known about primary resistance independent of BCR-ABL1 kinase activity. We reported a gene expression classifier of TKI-naïve CD34+ cells from chronic phase chronic myeloid leukemia (CP-CML) patients that predicts cytogenetic response to imatinib (McWeeney et al. Blood 2010). The expression signature associated with primary cytogenetic failure showed overlap with previously reported signatures of blast phase CML (BP-CML), suggesting that primary TKI resistance and advanced disease are biologically similar.Results: To identify critical genes involved in primary TKI resistance, we performed principal component analysis on the expression signature and identified the hematopoietic cell cycle regulator, MS4A3, as a key factor within this classifier. Importantly, low MS4A3 expression not only correlated with primary TKI resistance, but also with shorter overall survival (p<0.01), prompting us to study the role of MS4A3 in more detail. Since the signatures for primary cytogenetic failure and BP-CML overlapped, we next assessed whether MS4A3 expression was downregulated in BP-CML versus CP-CML. qRT-PCR confirmed that MS4A3 mRNA levels are markedly reduced (by >92%) in CD34+ cells from BP-CML patients (n=17; p<0.01) versus CP-CML patients (n=23) and normal controls (n=3). There was no significant difference between normal and chronic phase CD34+ cells, suggesting that MS4A3 is specifically downregulated upon CML blastic transformation. Microarray data from Oehler et al. (unpublished observations) and Zheng et al. (Leukemia 2006) also showed that MS4A3 mRNA levels are reduced in CD34+ cells from BP-CML patients (n=16; p<0.001) compared to CP-CML patients (n=19). Immunoblot analyses confirmed that MS4A3 protein was detectable in CD34+ cells from newly diagnosed CP-CML patients, but not in samples from patients with primary resistance who failed multiple TKIs but lack clinically relevant BCR-ABL1 mutations. Finally, in CP-CML samples from newly diagnosed patients, MS4A3 mRNA levels are 12-fold reduced in primitive CD34+38- stem cells compared to more committed CD34+38+ progenitor cells (n=3; p<0.05), which are innately resistant to BCR-ABL1 inhibition. Consistent with BCR-ABL1 kinase independence, MS4A3 mRNA and protein levels were unaffected by ex vivo TKI treatment.MS4A3 expression is also low in BP-CML cell lines, including K562, KYO-1, BV-173, KCL-22, and KU-812, with the notable exception of LAMA-84 cells. Thus, to understand the functional role of MS4A3 for TKI resistance, we introduced a doxycycline-inducible shRNA targeting MS4A3 (shMS4A3) into LAMA-84 cells. qRT-PCR confirmed 50-90% MS4A3 knockdown in the presence of doxycycline (0.1 µg/mL). Consistent with its role as a tumor suppressor, MTS assays revealed that MS4A3 knockdown increased the imatinib IC50 (n=3; p<0.05) and abolished the effects of imatinib in colony formation assays (n=2; p<0.05). We next assessed the effects of shMS4A3 in CD34+ cells from newly diagnosed CP-CML patients. qRT-PCR confirmed ~50% MS4A3 knockdown in primary cells. Despite the incomplete knockdown, shMS4A3 enhanced colony formation in the presence of imatinib in a dose-dependent manner (n=4; p<0.00001), and abolished imatinib-induced apoptosis (n=3; p<0.001). We also assessed the effects of ectopic MS4A3 expression in CD34+ cells from advanced phase CML (AP-CML) patients. qRT-PCR confirmed >2-fold upregulation of MS4A3. As expected, ectopic MS4A3 reduced colony formation by 55% in AP-CML (n=2; p<0.01), and enhanced sensitivity to imatinib-induced apoptosis by 35% (n=2; p<0.01). Neither shMS4A3 nor ectopic MS4A3 had any effect on survival of normal cord blood CD34+ cells (n=2).Conclusion: Our results suggest that MS4A3 is a tumor suppressor protein in CML that governs TKI responsiveness and is regulated in a BCR-ABL1 kinase-independent manner. MS4A3 loss confers TKI resistance to CP-CML patients destined to exhibit primary cytogenetic failure, and in BP-CML patients with refractory resistance. MS4A3 may also contribute to the innate resistance of primitive CML stem cells. Studies to identify the mechanism of MS4A3 downregulation in TKI resistance and how its loss biochemically impairs TKI response is currently underway and will be reported. DisclosuresAgarwal:CTI BioPharma: Research Funding. Deininger:BMS: Other: Consulting & Advisory Role, Research Funding; Novartis: Other: Consulting or Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role.

Abstract LB-020: Characterization of a novel BRAF in-frame deletion reveals a distinct mutational activating mechanism for oncogenic kinases

Abstract Kinase domain mutations are frequent drivers of many different types of cancer. While extensive studies of kinases such as BRAF and EGFR have provided insight into the mechanistic basis for many oncogenic point mutations such as the commonly occurring BRAFV600E and EGFRL858R mutations, the activation mechanism of short, in-frame deletions (such as the common EGFR exon19 deletion in non-small cell lung cancer) remains to be fully understood. In this work, we have discovered the activation mechanism for novel in-frame deletions in BRAF that have been reported in a small subset of thyroid cancers (TCGA Thyroid data set). These BRAF deletions structurally align with the EGFR exon19 deletion (most common being ΔELREA) and a similar deletion recently identified in HER2-positive breast tumor samples (ΔLRENT). These deletions target the β3-αC loop within the N-lobe of the kinase domain, which provides essential flexibility to the αC-Helix allowing the kinase to toggle between inactive and active conformations. Using molecular modeling studies, we demonstrate that all of these deletions genetically compromise this flexibility and constitutively “snap” the αC-Helix in the active conformation. Further characterization of the BRAF deletions shows that similar to BRAFV600E mutations, the β3-αC deletions are kinase-activating, CRAF-independent, and confer dimer-independent activity. Interestingly, cells with this deletion are sensitive to BRAF kinase inhibitors that bind to the active conformation (such as GDC-0879), but are innately resistant to the BRAF kinase inhibitor vemurafenib (which binds BRAF in the αC-Helix inactive, outward-shifted conformation). Similarly, modeling predicts all known β3-αC deletions would confer innate resistance to αC-Helix outward-shifting drugs such as vemurafenib or lapatinib. Further, we speculate that a β3-αC deletion could confer acquired resistance in vemurafenib-treated or lapatanib-treated patients. While many of the more common oncogenic point mutations have been suggested to function in part by destabilizing the inactive conformation and hence promoting the active conformation, it is becoming increasingly clear that oncogenic events that alter the αC-Helix region to directly promote the active conformation provide an alternative mechanism of kinase activation in cancer. Taken together our work underscores the importance of conformation specific kinase inhibitors to target mutationally activated kinases in cancer. Citation Format: Scott Foster, Aysegul Ozen, Dan Whalen, Kyung Song, Georgia Hatzivassiliou, Sarah Hymowitz, Nick Skelton, Shiva Malek. Characterization of a novel BRAF in-frame deletion reveals a distinct mutational activating mechanism for oncogenic kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-020. doi:10.1158/1538-7445.AM2015-LB-020

A new dehydratase conferring innate resistance to thiacetazone and intra-amoebal survival of Mycobacterium smegmatis.

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second-line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon-mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100-fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot-dog domains characteristic of dehydratase-like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis-Acanthamoeba co-cultures, we found that intra-amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.

P1-12-06: The Role of MAPK and PI3K/AKT/mTOR Signaling in Innate Lapatinib Resistance.

Abstract Background: Lapatinib is a tyrosine kinase inhibitor which blocks downstream MAPK and P13K/AKT/mTOR proliferation and survival signaling pathways in HER2 positive breast cancer cell lines, tumor xenografts and HER2 positive breast cancer patients. However, pre-clinical and clinical studies have shown that not all HER2 positive patients respond to lapatinib and thus innate resistance to lapatinib exists. The identification of biomarkers of lapatinib response is therefore critical and would enable individual therapeutic decisions to be based on tumor biology rather than basic histopathology data alone. The aim of this study was to examine the role of MAPK and PI3K/AKT/mTOR signaling in a panel of lapatinib sensitive and resistant HER2−amplified breast cancer cell lines to identify pharmacodynamic markers of response to lapatinib treatment. Methods: Dose response curves were generated to determine sensitivity to lapatinib in a panel of 17 HER2−amplified breast cancer cell lines. Total and phosphorylated levels of HER2, HER3, EGFR, AKT, ERK, S6K and eEF2 were determined following 24 hours lapatinib treatment in each of the cell lines. Results: Twelve of the cell lines were sensitive to lapatinib with IC50g &amp;lt; 1 μM while 5 of the cell lines were innately resistant to lapatinib with IC50g &amp;gt; 1 μM. Levels of pHER2 and pHER3 were decreased in response to lapatinib in all cell lines independent of sensitivity to lapatinib. This suggests that inhibition of HER2/HER3 activation is not indicative of response to lapatinib treatment. There was also no correlation between the levels of HER2, HER3, and EGFR and sensitivity to lapatinib in the cell line panel. In lapatinib sensitive cell lines, lapatinib decreased PI3K (pAKT), mTOR (pS6K) and MAPK (pERK) signaling and increased peEF2 levels. In contrast the levels of pAKT, pS6K, peEF2 and pERK were maintained following lapatinib treatment in lapatinib resistant cells. The continued activation of these proteins in the presence of lapatinib suggests a possible feedback mechanism that warrants further investigation. These data confirm that maintained signaling through either the P13K/AKT/mTOR pathway or the MAPK pathways in the presence of lapatinib can be an early pharmacodynamic biomarker of response. Conclusions: Maintenance of pAKT, pS6K, peEF2 and pERK levels, in response to lapatinib treatment correlates with lapatinib resistance. These data suggest that alterations in the P13K/AKT/mTOR and MAPK pathways play an important role in innate lapatinib resistance and pharmacologically targeting these pathways is a rational therapeutic approach for overcoming innate lapatinib resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-06.

Abc1p Is a Multidrug Efflux Transporter That Tips the Balance in Favor of Innate Azole Resistance in Candida krusei

Most Candida krusei strains are innately resistant to fluconazole (FLC) and can cause breakthrough candidemia in immunocompromised individuals receiving long-term prophylactic FLC treatment. Although the azole drug target, Erg11p, of C. krusei has a relatively low affinity for FLC, drug efflux pumps are also believed to be involved in its innate FLC resistance. We describe here the isolation and characterization of Abc1p, a constitutively expressed multidrug efflux pump, and investigate ERG11 and ABC1 expression in C. krusei. Examination of the ERG11 promoter revealed a conserved azole responsive element that has been shown to be necessary for the transcription factor Upc2p mediated upregulation by azoles in related yeast. Extensive cloning and sequencing identified three distinct ERG11 alleles in one of two C. krusei strains. Functional overexpression of ERG11 and ABC1 in Saccharomyces cerevisiae conferred high levels of resistance to azoles and a range of unrelated Abc1p pump substrates, while small molecule inhibitors of Abc1p chemosensitized C. krusei to azole antifungals. Our data show that despite the presence of multiple alleles of ERG11 in some, likely aneuploid, C. krusei strains, it is mainly the low affinity of Erg11p for FLC, together with the constitutive but low level of expression of the multidrug efflux pump Abc1p, that are responsible for the innate FLC resistance of C. krusei.

Gene Expression Profile of Chronic Myeloid Leukemia Innately Resistant to Imatinib

BackgroundAfter 12 months of treatment, most patients with chronic myeloid leukemia (CML) who receive imatinib as first-line therapy will have complete cytogenetic and molecular response. However, several patients will not exhibit molecular response, but their innate mechanism(s) of resistance remain poorly understood. We explored the molecular events involved in innate resistance in CML. Patients and MethodsFive patients who were molecular nonresponders and 7 major responders were investigated by using the expression profile of a set of 380 genes. Multiple testing procedure, Significance Analysis of Microarrays, Empirical Bayes Analysis of Microarrays, False Discovery Rate, and support vector machine with linear kernel were used for comparative analysis. ResultsThree hundred twenty-three of 380 genes (85%) were overexpressed in the nonresponder group compared with the responders. After a very stringent statistical analysis, a list of 26 genes was identified, in which overexpression in nonresponders was highly statistically significant. These genes are involved in signal transduction and transcription factors, apoptosis, cell cycle, and adhesion. Discriminative power of the proposed gene set was estimated by 2 different statistical methods, which yielded a correct prediction of the drug response for each patient used as a test sample. ConclusionOur study identified a set of 26 genes involved in resistance to imatinib, which can be used as clinical predictors or therapeutic targets. We consider some of them of particular interest: IGFBP2, CDC37, MAPK3, ETS1, and PEA15. Epigenetic mechanisms, such as genome-wide promoter hypomethylation, might be involved as the basic mechanism for innate resistance in CML.

Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate

Objective Resistance to imatinib mesylate monotherapy is clearly a barrier to successful treatment of chronic myeloid leukemia (CML) patients. In some patients, resistance arises due to powerful selective pressure on rare cells that carry amplified copies of the BCR-ABL fusion oncogene or point mutations in the Bcr-Abl tyrosine kinase domain that affect binding of the drug to the oncoprotein. However, in a proportion of patients neither mechanism operates, and resistance appears to be a priori, existing prior to exposure to the drug. These mechanisms of imatinib resistance are poorly understood and may be heterogeneous. Materials and methods We have previously described such innate resistance to imatinib in subclones of a myeloid leukemia cell line, KCL22, in which imatinib exposure inhibits the activity of Bcr-Abl and yet fails to induce apoptosis. We describe here whole-genome expression analysis of imatinib-sensitive and -resistant cells derived from the original KCL22 line, using Affymetrix microarray analysis. Results We detected differential expression of 39 genes that correlate with the imatinib-resistant phenotype. The resistant cells overexpress several genes associated with the suppression of apoptosis or with conferral of a transformed phenotype. Conclusion Amongst the differentially-expressed genes correlating with imatinib resistance, several suggest the activation of alternative pathway(s) that maintain viability and growth independently of Bcr-Abl kinase activity. Given the high rate of primary imatinib resistance in blast crisis, the potential of activating such alternative pathways appears to correlate with disease progression.

The study of innate drug resistance of human hepatocellular carcinoma Bel7402 cell line

The innate drug resistance of human hepatocellular carcinoma (HCC) Bel7402 cell line was studied in vitro. MTT assay showed that Bel7402 cells were innately resistant to doxorubicin (Dox), and even more resistant to vincristine (VCR). This resistance could be effectively reversed by verapamil (Ver), one of the classical multidrug resistance (MDR) modulating agents. However, the differences in 5-fluorouracil (5-FU) toxicity between these two cell lines is much less and the resistance of Bel7402 cells could only be slightly reversed by Ver, which may be experimental noise. Immunocytochemical staining using anti-p-glycoprotein monoclonal antibody JSB-1 indicated that the expression of the P-glycoprotein (P-gp) in the innate Bel7402 cells was elevated compared with the sensitive KB cells. The accumulation of Dox in innate resistant Bel7402 cells was 50.7% lower than that in sensitive KB cells by using spectrofluometric analyses, and the accumulation of Dox increased 1.6 fold in Bel7402 cells in the presence of Ver. The susceptibility of Dox-induced apoptosis was also increased in the presence of Ver by using flow cytometric assay and DNA fragmentation quantitative assay as well as by Hoechst 33258 staining. It appears that the innate Bel7402 cells might be useful in screening new antitumour drugs or new chemosensitisers which could overcome the innate or acquired resistant mechanism, and the toxicity and reversal effects with 5-FU are different from those known to be P-gp substrates such as VCR, Dox, and taxol.

Innate resistance to lethal mousepox is genetically linked to the NK gene complex on chromosome 6 and correlates with early restriction of virus replication by cells with an NK phenotype

Most inbred strains of mice, including DBA/2 (D2), are highly susceptible to the lethal effects of ectromelia virus, but C57BL/6 (B6) mice are innately resistant. Resistance is controlled by multiple, unlinked, autosomal dominant genes. Of 101 male (B6 x D2)F1 x D2 backcrossed (N2) mice, 18 died after ectromelia virus challenge and all were homozygous for the D2 allele at the proline-rich protein (Prp) locus on distal chromosome 6 (P < 0.001). This association was suggested by the patterns of susceptibility to lethal mousepox in recombinant inbred strains derived from B6 and D2 mice (D. G. Brownstein, P. N. Bhatt, L. Gras, and R. O. Jacoby, J. Virol. 65:1946-1951, 1991). The association between the Prp locus and susceptibility to lethal mousepox also held for N2 male mice that were castrated as neonates, which increased the percentage that were susceptible to 40. Spleen virus titers were significantly augmented in B6 (NK1.1+) mice depleted of asialo GM1+ or NK1.1+ cells, whereas spleen virus titers were unaffected in D2 (NK1.1-) mice depleted of asialo GM1+ cells. These results suggest that a gene or genes within the natural killer gene complex, adjacent to the Prp locus, determine strain variations in resistance to lethal ectromelia virus infection.

Activation of the hypothalamic-pituitary-adrenal axis differentially affects the anti-mycobacterial activity of macrophages from BCG-resistant and susceptible mice

The effect of hypothalamic-pituitary-adrenal (HPA) axis activation and exogenous glucocorticoids on the ability of splenic macrophages to control the growth of Mycobacterium avium was evaluated. We found that activation of the HPA axis by restraint stress or the addition of corticosterone increased the susceptibility of macrophages from mice that are innately susceptible to the in vivo growth of M. avium. In contrast, the ability of macrophages from innately resistant, congenic mice to control the growth of M. avium was not affected by HPA activation or the addition of corticosterone. The effect of restraint and of corticosterone on macrophage function was abrogated by either treating mice with the glucocorticoid receptor antagonist RU486 or the addition of the drug to cultures of macrophages. Activation of the HPA axis as well as the addition of corticosterone to cultures of macrophages resulted in a suppression of the production of tumor necrosis factor (TNF)-α and of reactive nitrogen intermediates by macrophages from both strains of mice. The lack of effect of HPA activation and of corticosterone on the mycobacterial resistance of macrophages from BCG-resistant mice, while at the same time suppressing the production of reactive nitrogen intermediates, appears to rule out a role for this antimicrobial pathway in innate resistance to mycobacterial growth.

Immunological consequences of innate resistance and susceptibility to BCG

The immunological consequences of genetically controlled innate resistance and susceptibility to Mycobacterium bovis (BCG) infection in mice were investigated. The susceptible ( Bcg s) BALB/c and the congenic resistant BALB/c. Bcg r mouse strains were employed to test for differences in the specific immune response to BCG. Antigen-specific lymphocyte proliferation and production of interleukin 2 (IL-2) to purified protein derivative (PPD) in vitro were measured following infection of congenic mice with low (10 4) and high (10 6) doses of BCG. The lymphocyte subsets were identified by testing the ability of spleen cells to respond after separation of T and B cells and after cytotoxic depletion of T cell subsets. In addition, fluorescence activated cell sorter (FACS) analysis with anti-T and -B cell monoclonal reagents was used to enumerate lymphocyte populations in BCG-infected spleens. The results indicated that the innately resistant mice displayed antigen-specific T helper cell function three weeks following the injection of BCG. The susceptible animals were found to be T cell-unresponsive since they lacked both proliferative and IL-2 secreting specific T cells. No evidence for suppression of IL-2 production or proliferation was detected in BALB/c (susceptible) spleen cultures in cell mixing experiments. The results provide evidence for a regulatory role of the Bcg gene on the generation of lymphocyte responses to BCG.

Heterologous synergistic interactions in concurrent experimental infection in the mouse with Schistosoma mansoni, Echinostoma revolutum, Plasmodium yoelii, Babesia microti, and Trypanosoma brucei.

Primary infections with Plasmodium yoelii and Echinostoma revolutum in the mouse induced a significant increase in the heterologous Schistosoma mansoni challenge worm establishment, whereas S. mansoni worm establishment remained unaffected by primary infections with Trypanosoma brucei and Babesia microti. Concurrent infection in the mouse with P. yoelii or T. brucei, but not with B. microti, blocked the resistance to homologous E. revolutum challenge infection, and primary P. yoelii and T. brucei infections and corticosteroid treatment made naive, innately resistant mice susceptible to E. revolutum infection. Innate resistance to infection with E. revolutum, the pattern of expulsion of low-level E. revolutum infections, and resistance to homologous S. mansoni challenge infection remained unaffected by concurrent B. microti infection. Primary, heavy E. revolutum infections in the mouse resulted in the enhancement of subsequent infection with B. microti, whereas primary infection with S. mansoni suppressed subsequent B. microti infection in some but not all experiments. In a single experiment, P. yoelii infection was suppressed markedly by primary S. mansoni infection, whereas the enhancement of P. yoelii infection in concurrently E. revolutum-infected mice was seen in only one of the several experiments conducted. However, no interference with resistance to homologous B. microti and P. yoelii challenge infection was induced by concurrent infection with S. mansoni and E. revolutum. We suggest that the synergistic interactions demonstrated between protozoans and helminths in concurrent experimental infection in the mouse are induced by immunosuppression.