Drug-resistant Clones Research Articles

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae.

Klebsiella pneumoniae has emerged as an important cause of two distinct public health threats: multi-drug resistant (MDR) healthcare-associated infections and drug susceptible community-acquired invasive infections. These pathotypes are generally associated with two distinct subsets of K. pneumoniae lineages or ‘clones’ that are distinguished by the presence of acquired resistance genes and several key virulence loci. Genomic evolutionary analyses of the most notorious MDR and invasive community-associated (‘hypervirulent’) clones indicate differences in terms of chromosomal recombination dynamics and capsule polysaccharide diversity, but it remains unclear if these differences represent generalised trends. Here we leverage a collection of >2200 K. pneumoniae genomes to identify 28 common clones (n ≥ 10 genomes each), and perform the first genomic evolutionary comparison. Eight MDR and 6 hypervirulent clones were identified on the basis of acquired resistance and virulence gene prevalence. Chromosomal recombination, surface polysaccharide locus diversity, pan-genome, plasmid and phage dynamics were characterised and compared. The data showed that MDR clones were highly diverse, with frequent chromosomal recombination generating extensive surface polysaccharide locus diversity. Additional pan-genome diversity was driven by frequent acquisition/loss of both plasmids and phage. In contrast, chromosomal recombination was rare in the hypervirulent clones, which also showed a significant reduction in pan-genome diversity, largely driven by a reduction in plasmid diversity. Hence the data indicate that hypervirulent clones may be subject to some sort of constraint for horizontal gene transfer that does not apply to the MDR clones. Our findings are relevant for understanding the risk of emergence of individual K. pneumoniae strains carrying both virulence and acquired resistance genes, which have been increasingly reported and cause highly virulent infections that are extremely difficult to treat. Specifically, our data indicate that MDR clones pose the greatest risk, because they are more likely to acquire virulence genes than hypervirulent clones are to acquire resistance genes.

Klebsiella pneumoniae ST307 with blaOXA-181, South Africa, 2014-2016.

Klebsiella pneumoniae sequence type (ST) 307 is an emerging global antimicrobial drug–resistant clone. We used whole-genome sequencing and PCR to characterize K. pneumoniae ST307 with oxacillinase (OXA) 181 carbapenemase across several private hospitals in South Africa during 2014–2016. The South Africa ST307 belonged to a different clade (clade VI) with unique genomic characteristics when compared with global ST307 (clades I–V). Bayesian evolution analysis showed that clade VI emerged around March 2013 in Gauteng Province, South Africa, and then evolved during 2014 into 2 distinct lineages. K. pneumoniae ST307 clade VI with OXA-181 disseminated over a 15-month period within 42 hospitals in 23 cities across 6 northeastern provinces, affecting 350 patients. The rapid expansion of ST307 was most likely due to intrahospital, interhospital, intercity, and interprovince movements of patients. This study highlights the importance of molecular surveillance for tracking emerging antimicrobial clones.

Detection in the United Kingdom of the Neisseria gonorrhoeae FC428 clone, with ceftriaxone resistance and intermediate resistance to azithromycin, October to December 2018.

We describe detection in the United Kingdom (UK) of the drug-resistant Neisseria gonorrhoeae FC428 clone, with ceftriaxone resistance and intermediate azithromycin resistance. Two female patients developed infection following contact with UK-resident men from the same sexual network linked to travel to Ibiza, Spain. One case failed treatment with ceftriaxone, and azithromycin and gentamicin, before successful treatment with ertapenem. Both isolates had indistinguishable whole-genome sequences. Urgent action is essential to contain this drug-resistant strain.

Interleukin-6 Interweaves the Bone Marrow Microenvironment, Bone Loss, and Multiple Myeloma.

The immune system is strongly linked to the maintenance of healthy bone. Inflammatory cytokines, specifically, are crucial to skeletal homeostasis and any dysregulation can result in detrimental health complications. Interleukins, such as interleukin 6 (IL-6), act as osteoclast differentiation modulators and as such, must be carefully monitored and regulated. IL-6 encourages osteoclastogenesis when bound to progenitors and can cause excessive osteoclastic activity and osteolysis when overly abundant. Numerous bone diseases are tied to IL-6 overexpression, including rheumatoid arthritis, osteoporosis, and bone-metastatic cancers. In the latter, IL-6 can be released with growth factors into the bone marrow microenvironment (BMM) during osteolysis from bone matrix or from cancer cells and osteoblasts in an inflammatory response to cancer cells. Thus, IL-6 helps create an ideal microenvironment for oncogenesis and metastasis. Multiple myeloma (MM) is a blood cancer that homes to the BMM and is strongly tied to overexpression of IL-6 and bone loss. The roles of IL-6 in the progression of MM are discussed in this review, including roles in bone homing, cancer-associated bone loss, disease progression and drug resistance. MM disease progression often includes the development of drug-resistant clones, and patients commonly struggle with reoccurrence. As such, therapeutics that specifically target the microenvironment, rather than the cancer itself, are ideal and IL-6, and its myriad of downstream signaling partners, are model targets. Lastly, current and potential therapeutic interventions involving IL-6 and connected signaling molecules are discussed in this review.

Acinetobacter baumannii infections in Amazon Region driven by extensively drug resistant international clones, 2016-2018.

BACKGROUND Acinetobacter baumannii is a leading cause of nosocomial infections. This species is characterised by the presence of pandemic lineages (International Clones) that present a broad antimicrobial resistance profile.OBJECTIVE To perform the molecular epidemiology of carbapenem-resistant A. baumannii from a clinical setting in the Amazon Basin, and to characterise their antimicrobial resistance determinants.METHODS The genetic relationship of carbapenem-resistant A. baumannii were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Class A, B and D β-lactamase genes were screened by polymerase chain reaction (PCR) and sequencing. The antimicrobial susceptibility profile was obtained by Disc-diffusion method and minimum inhibitory concentration (MIC) determination.FINDINGS All carbapenem-resistant A. baumannii strains belonged to three international clones, IC-1, IC-5 and IC-6, the latter recently reported by the first time in Brazil. The major determinant of carbapenem resistance in IC-1 and IC-5 strains was bla OXA-23, associated with ISAba1 and ISAba3, respectively, while IC-6 harboured the bla OXA-72.CONCLUSIONS The A. baumannii epidemiology in Brazilian Amazon Region was unknown. It was demonstrated that A. baumannii XDR international clones were responsible for nosocomial infections in Boa Vista during 2016-2018, revealing that the epidemiological scenario of A. baumannii infections in Amazon Region resembles those from the cosmopolitan regions worldwide.

Exome Sequencing of Drug-Resistant Clones for Target Identification.

Many small molecule compounds with anticancer activities are discovered through phenotype-based screens. However, discovering the targets of these small molecules has been challenging. The gold standard for target identification requires the discovery of mutations in the target protein that block the effects of small molecules in vitro as well as in vivo. Here we describe the procedures for isolating drug resistant clones using the colorectal cancer cell line HCT-116 followed by whole-exome sequencing to identify recurrent mutations associated with compound resistance. Together with downstream in vitro and in vivo validation experiments, this strategy enables rapid target discovery for cytotoxic compounds.

Line-probe assay and molecular typing reveal a potential drug resistant clone of Mycobacterium tuberculosis in Ethiopia

BackgroundAntimicrobial resistance is a global concern of increasing significance. Multidrug resistant tuberculosis (MDR-TB) is spreading worldwide. It is important to monitor trends of antimycobacterial resistance. This is particularly true for high TB burden countries such as Ethiopia where disproportionally less drug sensitivity data are reported from.MethodsThe prevalence of drug resistance was assessed with the line probe assay GenoType MTBDRplus in a set of 161 M. tuberculosis strains that were selected from four common lineages and sub-lineages previously identified in Ethiopia. Most of the tested M. tuberculosis isolates had been genotyped by established Spoligotyping and MIRU-VNTR typing methods.ResultsThe proportion of MDR-TB among the isolates was 3.1%. Mono-resistance was 1.2% to rifampicin and 4.3% to isoniazid, and resistance to either of the two first line drugs was 8.7%. Strains of Lineage 4 had the highest resistance rate (13.6%) followed by Lineage 3 (4.9%). None of the isolates representing Lineages 1 and Lineage 7 were drug resistant. Multidrug resistance among pulmonary TB and TB lymphadenitis clinical isolates was 2.8 and 3.7%, respectively. Drug resistance of strains carrying the most prevalent spoligotype in Ethiopia - SIT149 - was further explored. Stratification by MIRU-VNTR identified one genotype with a high rate of drug resistance against Rifampicin and Isoniazid and circulation of a potential MDR-TB clone is proposed.ConclusionAlthough the strain selection was not fully randomized, the overall M. tuberculosis drug resistance rate in this strain set was 8.7% while the rate of MDR was 3.1%. In parallel, we identified a sub-lineage that showed a high rate of resistance to both rifampicin and isoniazid. These resistant strains may belong to a clone of M. tuberculosis that is circulating in the highlands of Ethiopia.

Mutational Landscape and Temporal Evolution during Treatment of Relapsed Acute Lymphoblastic Leukemia

▪IntroductionRelapsed childhood acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and has poor prognosis due to acquired drug resistance. However, the clonal evolution leading to drug resistance at ALL relapse is incompletely understood.MethodsWe performed whole-genome sequencing (WGS) of samples at diagnosis and relapse from 103 Chinese patients, most of whom were enrolled on the Shanghai Children's Medical Center (SCMC) ALL2005 frontline treatment protocol. We also performed ultra-deep sequencing at 5,000-50,000X coverage of 211 serial bone-marrow samples from 17 of these patients (3-23 per case) collected during ALL therapy. Fifteen ALL subtypes were identified based on copy number variation, structural re-arrangement and gene fusion by WGS and RNA-Seq analysis. Cases were selected based on sample availability with no bias on age, gender, cytogenetics, or immunophenotype compared to the characteristics of all relapsed cases at SCMC; however only one CRLF2-rearranged case was observed suggesting that CRLF2-rearrangement could be a rare event in Asian patients. Functional impact of novel mutations was assessed by ectopic expression in ALL cell lines.ResultsRelapse-specific somatic alterations were significantly enriched in 12 genes (NT5C2, NR3C1/2, PRPS1/2, TP53, CREBBP, MSH2/6, PMS2, WHSC1, and FPGS) predominantly involved in drug metabolism and response pathways. These somatic alterations were present in over 50% (56/103) of relapsed ALLs and were enriched in patients with early relapse (9-36 months from diagnosis) compared to very early (<9 months) or late relapse (>36 months) patients. NR3C1 mutations resulted in loss of glucocorticoid receptor transcriptional activity and severely impaired glucocorticoid response in vitro, while FPGS mutations led to reduced enzymatic activation of methotrexate. Genome-wide analysis identified 9 mutational signatures, including two novel ones exclusive to relapse. Novel signature 1 was characterized by C>G mutations and was present in 15% of the relapsed cases with an enrichment for hyperdiploid ALL, while novel signature 2, present in 14% of the relapsed cases, was characterized by C mutations (C>T in particular) followed by a G. These novel signatures gave rise to relapse-specific drug resistance mutations, including PRPS1, TP53, NT5C2, and KRAS mutations.The majority (59%) of the cases underwent a selective sweep as the relapse clone arose from a subclone detectable at diagnosis; however, very early relapse cases were less likely to experience a clonal sweep and had multiple lineages present at relapse. In patients tracked serially through multiple relapses, the clonal composition of drug resistance variants evolved substantially in response to chemotherapy. For example, in one patient, two independent NT5C2-mutant clones appeared at relapse, manifesting convergent evolution (Fig. 1a). One of the NT5C2-mutant clones expanded after subsequent treatment while the other diminished in prevalence. Sequential acquisition of multiple drug resistance mutations was also evident, with one patient sequentially acquiring KRAS, FBXW7, NR3C1 and FPGS mutations over a period of 5 years through multiple relapses (Fig. 1b). Indeed, 17% (18/103) of patients acquired multiple drug resistance mutations at relapse. The median time from detecting resistant clones to relapse was 41 days, suggesting that ultra-deep sequencing offers a means for early detection of genetic lesions that could serve as an indicator of relapse to enable earlier therapeutic intervention.ConclusionsVery early relapses likely have different resistance mechanisms than early or late relapses, perhaps due to increased intrinsic resistance or the presence of multiple drug-resistant clones. At least a subset of relapse-specific drug resistance mutations may be acquired de novo, rather than pre-existing, as evidenced by resistance mutations bearing the novel relapse-specific mutational signatures which are likely to be therapy-induced. Relapsed ALL can acquire multiple drug resistance mutations targeting different drug classes. Frequent monitoring of disease, such as via cell-free DNA sequencing, may enable earlier detection of relapse and facilitate timely treatment to avoid selection for drug-resistant clones. DisclosuresNo relevant conflicts of interest to declare.

A Patient Derived Xenograft As a Preclinical Model for Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a type of rare aggressive lymphoma accounting for 5.4% of primary intestinal peripheral T-cell lymphoma and 10-25% of all primary intestinal lymphoma. MEITL also has an extremely poor prognosis with the median overall survival of only seven months. No effective treatment or targeted therapies are currently available to manage this disease. Therefore, there is an urgent need to devise and establish an appropriate preclinical model for a better understanding of this disease and for new therapeutic strategies to be developed on it.Here, we present the first MEITL patient derived xenograft (PDX) as both orthotropic intestinal and as a subcutaneous model. The histological analysis demonstrated high similarity in the overall immunomorphologic features between the primary tumour and PDX tumours. Importantly, all the PDX tumors carried the distinctive MEITL immunophenotype; CD3+CD4-CD8+CD56+ and extensive nuclear expression of MATK. We also had Sanger sequenced 83 somatic mutations in the original tumour and rediscovered them in the 6th passage of our xenografts, which suggest that the generated MEITL PDX tumours were genetically stable.Moreover, whole exome sequencing results showed that the clonal architecture in the primary tumour was well preserved in the PDX tumours. The clonal architecture was further analysed with single cell whole genome amplification following by Sanger sequencing. We found that the most of heterozygous mutations in the primary tumour were a mixture of wild type alleles and heterozygous/homozygous mutations from separated clones, including DUPS14 heterozygous mutation (p.R300W) which was known to activate MEK pathway, and SETD2 mutations, (g.chr3:47061285_ 47061286insA and g.chr3:47127805C>A) which were the synthetic lethal partner of Wee1. Interestingly, we found that the two SETD2 mutations alone were able to differentiate three subclones. This result indicates that the tumour was highly subclonal, which could have a strong impact on the selection of drug resistant clones in the drug treatment.In order to verify this hypothesis, we proceeded to test the efficacy of MEK inhibitor, Trametinib and Wee1 inhibitor, AZD1775 both separately and combined treatment, in our PDX subcutaneous model. The results showed that both 1mg/kg/day of Trametinib and 60mg/kg/day of AZD1775 treatments significantly delayed 60-70% of tumour growth as compared with the vehicle group. The tumour growth was completely arrested in the combined treatment group. In addition, 20% and 40% of the mice exhibited Trametinib and AZD1775 resistance, respectively. From our genomic analysis, all DUPS14 and SETD2 mutations from the pretreated tumours were preserved in the treatment-resistant tumours but absent in the sensitive post-treated residual tumours. The clonality analysis from the sequencing data of the pretreated tumour supports and suggests that single-agent regimes will often not be able to induce a complete response in MEITL patients.Altogether, the current results demonstrated that our MEITL PDX model preserved the principal histological and genetic characteristics of its original tumour and is genomic stable across passages. The model also showed that MEITL could be highly subclonal and combined targeted therapy might achieve a higher efficacy for MEITL treatment. DisclosuresNo relevant conflicts of interest to declare.

The Gfi1-SphK1 Axis Regulates the Growth and Survival of Myeloma Cells

In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable hematologic malignancy due to emergence of drug-resistant clones. We previously reported that MM cells upregulate expression of the transcriptional repressor, Growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSC), which induces prolonged inhibition of osteoblast differentiation. We recently showed that Gfi1 levels are also increased in the majority of CD138+ cells from MM patients and cell lines ,and Gfi1 levels significantly correlated with Mcl-1 protein expression. Further, Gfi1 repressed MM cell death by inhibiting expression of apoptosis-inducing genes. Importantly, Gfi1 overexpression in MM cells enhanced MM cell growth and conferred resistance to proteasome-inhibitor- induced apoptosis. However, the mechanisms responsible for these effects of Gfi1are unknown.Sphingosine kinase 1 (SphK1) is overexpressed in many cancers including MM, and catalyzes the phosphorylation of sphingosine (SPH) to sphingosine-1-phosphate (S1P). S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells and angiogenesis, all of which contribute to cancer progression. Therefore, we hypothesized that adhesive interactions between MM cells and BMSC stimulate survival and growth of MM cells in part through the Gfi1-SphK1 axis by modulating their sphingolipid profile (Ceramide/SPH/S1P ratio).We found that SphK1 mRNA is highly expressed in CD138+ cells from MM patients and cell lines compared with normal donors. Further, Gfi1 protein expression correlated significantly with SphK1 protein level in CD138+ cells from MM patients and MM cell lines. Soluble factors (IL-6 and S1P) that are increased in the MM microenvironment, hypoxia (1% O2) and adhesive interactions of MM cells with BMSC further increased Gfi1 and SphK1 mRNA and protein levels in MM cells. Gfi1 Knockdown (KD) in MM cells induced a profound decrease of SphK1 mRNA and protein activity and inhibited MM cell growth and viability. In contrast, over-expression of Gfi1 in MM cells increased SphK1 levels that conferred a survival advantage to MM cells over empty vector-transduced control cells. Increased Gfi1 expression also resulted in increased intracellular S1P and decreased sphingosine levels, as measured by LC-MS/MS. Further, treatment of MM cells with a SphK1 specific inhibitor (SKI2), dose-dependently reduced MM cell viability at 24h, regardless of their p53 status. Interestingly, p53 null MM cells were more resistant to SK12, as compared with p53 replete cells. In p53 replete MM cells, SphK1 inhibition significantly reduced c-Myc protein expression, induced autophagy (as shown by increased LC3 II protein levels) and increased total ceramides levels. Moreover, BMSC protected p53 replete MM cells from the anti-survival effects of SKI2 in 3D cultures. These data suggest that Gfi1 regulates MM growth in part by enhancing the expression and activity of SphK1.Taken together, our results support that Gfi1 acts as a key regulator of MM growth and survival, at least partially through modulation of SphK1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. DisclosuresRoodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Loss of KDM6A Confers Drug Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic cancer resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy, relapse caused by intrinsic or acquired drug resistance remains a major hurdle in the treatment of AML. Recently, we found KDM6A as a novel relapse-associated gene in a cohorte of 50 cytogenetically normal AML patients. KDM6A (or UTX) is a histone 3 lysine 27 (H3K27)-specific demethylase and a member of the COMPASS (complex of proteins associated with Set1)-like complex, which is important for chromatin enhancer activation. KDM6A is targeted by inactivating mutations in a variety of cancer types with frequency of occurrence ranging from 0.7 to 4% in AML.In this study, we used matched diagnosis and relapse samples from AML patients, patient-derived xenografts (PDX), and myeloid leukemia cell lines to investigate the status of KDM6A during disease progression and the implications of KDM6A loss regarding chemotherapy resistance. We found three AML patients with enrichment of KDM6A mutations at relapse and mutation-independent, relapse-specific loss of KDM6A expression in three additional AML patients. KDM6A mutations comprise deletions and point mutations and appear to be mainly loss-of-function mutations. In addition, we examined the mutation profile and KDM6A expression in patient-derived xenograft (PDX) samples from 8 relapsed AML patients. In 4/8 samples, KDM6A protein levels were low or completely lost. Due to the fact that all patients had received induction therapy including single or combination treatment with agents such as cytarabine (AraC), daunorubicin (DNR), and 6-thioguanine (6-TG), we hypothesized that loss of KDM6A confers resistance to chemotherapy. To exclude gender-specific effects (KDM6A escapes X inactivation leading to higher levels in females), we compared male KDM6A knockout (KO) with WT leukemia cell lines and found increased AraC resistance in the KDM6A KO cells (unpaired, two-tailed Student's t-test; P=0.0441). In addition, we treated two relapsed PDX AML cells of the same gender, AML 491 (KDM6A WT and strong expression) and AML 393 (KDM6A mutation and weak expression) with AraC for 72h in vitro and found significantly increased AraC resistance in the KDM6A-mutant PDX AML 393 cells (P=0.016). To further investigate whether reduced expression or loss of KDM6A leads to increased resistance towards multiple drugs, we silenced KDM6A expression by shRNA or CRISPR/Cas9 in K562 and MM-1 cells. Compared to control, KDM6A knockdown (KD) and KO K562 cells showed a strong proliferative advantage after AraC and DNR but not 6-TG treatment. A similar drug resistance phenotype was observed in KDM6A KO MM-1 cells.To unravel the mechanism of drug resistance, we performed RNA-Seq analysis in K562 cells treated with siRNA or shRNA against KDM6A under native conditions and after AraC (150nM) treatment for 72h. We compared these differentially expressed genes with known key candidate genes in AraC, DNR, and 6-TG metabolic pathway and found that ENT1 was consistently downregulated in KDM6A KD cells in both siRNA- and shRNA-mediated RNA-Seq screenings. Decreased ENT1 levels were also detected in KDM6A KO K562 single cell clones. ENT1 (also known as SLC29A1) is a membrane transporter relevant for the cellular uptake of nucleosides and its analogues. Competitive inhibition of ENT1 by the small molecule antagonist NBMPR lead to decreased sensitivity towards AraC but not DNR and 6-TG suggesting that increased AraC resistance in KDM6A KO cells is caused, at least partially, by downregulation of ENT1.To elucidate the mechanism of ENT1 regulation by KDM6A, we performed ChIP-seq analysis for H3K27me3 and H3K27ac in the sister cell lines MM-1 (KDM6A WT) and MM-6 (KDM6A KO). ChIP-seq for H3K27me3 showed no enrichment on the ENT1 locus, but we detected differential H3K27ac peaks in the promoter and a putative enhancer region of ENT1 in MM-1 compared to MM-6. These data suggest that increased ENT1 expression may function through direct or indirect effects of KDM6A on enhancer regions, independent of its H3K27 demethylase activity.In conclusion, our results show that mutations in KDM6A are associated with the outgrowth of drug-resistant clones and highlight KDM6A as a novel biomarker of drug resistance in AML. DisclosuresHiddemann:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Growth factor independence 1 expression in myeloma cells enhances their growth, survival, and osteoclastogenesis

BackgroundIn spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown.MethodsHuman primary CD138+ and BMSC were purified from normal donors and MM patients’ bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo.ResultsWe found that Gfi1 expression is increased in patient’s MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth.ConclusionsThese results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM.

The AMPK inhibitor overcomes the paradoxical effect of RAF inhibitors through blocking phospho–Ser-621 in the C terminus of CRAF

The dimerization-driven paradoxical activation of RAF proto-oncogene Ser/Thr kinase (RAF) is the predominant cause of drug resistance and toxicity in cancer therapies with RAF inhibitors. The scaffold protein 14-3-3, which binds to the RAF C terminus, is essential for RAF activation under physiological conditions, but the molecular basis is unclear. Here we investigated whether and how 14-3-3 regulates the dimerization-driven transactivation of the RAF isoform CRAF by RAF inhibitors and affects drug resistance and toxicity by virtue of the dominant role of CRAF in these processes. We demonstrated that 14-3-3 enhances the dimerization-driven transactivation of CRAF by stabilizing CRAF dimers. Further, we identified AMP-activated protein kinase (AMPK) and CRAF itself as two putative kinases that redundantly phosphorylate CRAF's C terminus and thereby control its association with 14-3-3. Next, we determined whether the combinatory inhibition of AMPK and CRAF could overcome the paradoxical effect of RAF inhibitors. We found that the AMPK inhibitor (AMPKi) not only blocked the RAF inhibitor–driven paradoxical activation of ERK signaling and cellular overgrowth in Ras-mutated cancer cells by blocking phosphorylation of Ser-621 in CRAF but also reduced the formation of drug-resistant clones of BRAFV600E-mutated cancer cells. Last, we investigated whether 14-3-3 binding to the C terminus of CRAF is required for CRAF catalytic activity and observed that it was dispensable in vivo. Altogether, our study unravels the molecular mechanism by which 14-3-3 regulates dimerization-driven RAF activation and identified AMPKi as a potential agent to counteract drug resistance and adverse effects of RAF inhibitors in cancer therapies.

Abstract 4073: Using patient-derived PDAC cells to understand c-MYC involvement in the drug resistance mechanisms of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that is typically incurable due to acquired resistance to systemic chemotherapy. A deeper understanding of the mechanisms involving drug resistance is needed to improve patient outcomes. Commercially available PDAC cell lines are the most used model in research but do not accurately recapitulate a given patient's tumor. Using the conditional reprogrammed cell (CRC) technique, we established patient-specific cell lines and created nab-paclitaxel (nab-PTX)-resistant clones, which allowed us to better study resistance mechanisms. In many cancers, c-MYC has a known function in tumor progression, and recent studies suggest that the c-MYC pathway is involved in PDAC. However, the role of c-MYC in the mechanisms leading to nab-PTX resistance has not been extensively explored. Methods: Biopsies were collected from treatment-naive PDAC patients, and long-term cultures of PDAC CRCs were established. The PDAC origin of the cell lines was validated with genomic analysis. The IC50s for nab-PTX were calculated and used to generate drug-resistant clones. The Zebrafish model was used to verify the drug resistance in vivo, while subcutaneous injections of the parental and drug-resistant CRCs in nude mice were performed to test the cells' ability to form tumors. Exome sequencing, microarray, and Western blotting analyses were used to characterize and compare the parental lines with the drug-resistant clones and to identify key pathways involved in the evolution of drug resistance. Results: We established KRAS-mutant primary cell lines from PDAC patients' biopsies. Using 2 parental lines, we generated 5 nab-PTX-resistant clones, with IC50s up to 800 times greater than those of the matched parents; resistant profiles were verified in Zebrafish. Subcutaneous injections in nude mice showed the CRCs' ability to form tumors that recapitulate human PDAC. A greater metastatic phenotype was observed in the tumors derived from the resistant clones. RNA microarrays, comparing the parental cells with the resistant clones, identified a possible resistance mechanism involving the induction of a pro-inflammatory pathway leading to c-MYC overexpression. Western blotting, as well as immunocytochemical and immunohistochemical staining, performed on the CRCs and CRC-PDX-derived samples confirmed the role of c-MYC overexpression in the evolution of nab-PTX resistance. Conclusion: The conditional reprogrammed cell (CRC) methodology addresses the need for a reliable method for generating primary cell lines on a single patient basis. The ability to rapidly model in vitro, and verify in vivo, that the overexpression of c-MYC contributes to the development of nab-PTX resistance is a significant advancement in the field and provides a platform for discovery of more effective treatment of refractory PDAC. Citation Format: George S. Avetian, Erika M. Parasido, Jonathan Brody, Jordan Winter, Eric Londin, Michael Pishvaian, Eric Glasgow, Stephen Byers, Christopher Albanese. Using patient-derived PDAC cells to understand c-MYC involvement in the drug resistance mechanisms of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4073.

Abstract 791: CG'806, a first-in-class pan-FLT3/pan-BTK inhibitor, demonstrates superiority to other FLT3 and BTK inhibitors against primary patient samples

Abstract Introduction: While acute myeloid leukemia (AML) is a complex and heterogeneous malignancy, the most common mutation is the internal tandem duplication (ITD) of FLT3 occurring in ~30% of AML patients. Several FLT3 inhibitors have transient clinical benefit, lasting only 3-4 months, often due to emergence of drug resistant clones with additional mutations in FLT3. Thus, there is need for a pan-FLT3 inhibitor to control all mutant forms of FLT3, and to suppress diverse oncogenic clones. Likewise, overexpression of Bruton's tyrosine kinase (BTK) is a driver of chronic lymphocytic leukemia (CLL). Ibrutinib, a covalent BTK inhibitor approved for CLL and certain other B-cell malignancies, is limited by resistance resulting from mutation at cysteine residue 481 to serine (BTK-C481S), prompting a need for new agents to inhibit all forms of BTK. CG'806 is a small molecule that inhibits wild type (WT) FLT3, FLT3-ITD and tyrosine kinase domain (TKD) point mutations, including D835G, D835Y, D835H, F691L; it also inhibits BTK WT and BTK-C481S. We profiled CG'806 against primary samples from patients with AML, CLL and other hematologic malignancies to determine its activity and potency relative to other FLT3 inhibitors. CG'806 and ibrutinib were compared directly for sensitivities on primary CLL samples and various B-cell and other hematologic malignancies. Methods: Inhibitor activity was assessed by an ex vivo assay to determine sensitivities of CG'806 and other FLT3 and BTK inhibitors on freshly isolated primary patient samples. Cell viability was assessed after 72-hour culture using a tetrazolium-based MTS assay and IC50 values calculated as a measure of drug sensitivity. For AML samples, mutational status of ITD and TKD in FLT3 were assessed by a PCR assay for ITD and by whole exome sequencing for TKD. Results: CG'806 has more potent activity against a broader subset of AML samples relative to other FLT3 inhibitors, including midostaurin, gilteritinib, quizartinib, sorafenib, crenolanib, and dovitinib. This was especially true in FLT3-ITD and FLT3-TKD positive cases, although enhanced activity was also observed in FLT3 WT samples. CG'806 potency correlated with FLT3-ITD allele frequency as cases with higher allelic frequency generally had greater sensitivity in comparison to lower allelic frequency cases. CG'806 had greater activity on primary CLL samples than ibrutinib, which may be due to the dual activity of CG'806 against CSF1R (FMS) (IC50 = 0.6nM), a recently described target in CLL that provides a pro-tumor signal from nurse-like monocyte/macrophage lineage cells. Conclusions: CG'806 is a potent and broadly active FLT3 inhibitor in AML and BTK inhibitor in CLL. These data reveal CG'806's enhanced activity relative to kinase inhibitors currently approved for each indication and support further clinical development of CG'806 for both AML and CLL. Citation Format: Stephen E. Kurtz, Kevin Watanabe-Smith, Dan Bottomly, Beth Wilmot, Shannon Mcweeney, Andrea Local, Hongying Zhang, Stephen Howell, William Rice, Brian J. Druker, Jeffrey W. Tyner. CG'806, a first-in-class pan-FLT3/pan-BTK inhibitor, demonstrates superiority to other FLT3 and BTK inhibitors against primary patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 791.

2 Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

IntroductionClinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.Material and methodsExome analysis was performed on a panel of CRC cells that became resistant to commonly used targeted agents to uncover genetic alterations acquired under drug selective pressure. Tumour evolution of resistant cell population was then inferred through EXPANDs software. β-catenin activity, cell growth and death were measured upon functional and pharmacological modulation of WNT pathway in resistant cells and preclinical models established from tissues biopsy obtained from CRC patients who relapsed during treatment with targeted therapies.Results and discussionsExome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, each associated with distinct genomic landscapes. Functional and pharmacological inhibition of WNT signalling induces cell death in CRC preclinical models, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT pathway resulted in translocation of β-catenin to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones and prevent onset of secondary resistance.ConclusionIn summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumours face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments.

PO-490 Treatment of P53 wild-type cells with P53-inducer idasanutlin® (RG7388) generates stable drug-resistant clones

IntroductionThe protein p53 is activated in response to DNA damage. The active protein induces cell cycle arrest and DNA repair program and thus protects the organism against carcinogenic events. Inactivation of p53 gives the cancer cell a profit of genetic instability, resulting in enhanced cancer cell microevolution. Apart from gaining direct mutations in p53-coding gene, overexpression of Mdm2 protein, which is a natural negative regulator of p53, is the most common method utilised by cancer to keep p53 inactive. Mdm2 binds to p53, blocks its transactivation domain and directs p53 to degradation. The re-activation of p53 protein by forced dissociation of Mdm2-p53 complexes using small molecule Mdm2 antagonists is currently believed to be a promising therapeutic strategy of the treatment of cancers expressing wild-type p53. Several representatives of this class of molecules, such as Idasanutlin (RG7388), are currently evaluated in clinical trials. However no data concerning the generation of secondary resistance is available for those modern, optimised compounds.Material and methodsHuman osteosarcoma U-2 OS cells were treated with Idasanutlin, followed by the analysis of viability (MTT assay), p53 activation (western blotting), the induction of apoptosis (annexin V staining) and cell cycle distribution (double staining with BrdU/FITC-anti-BrdU and propidium iodide). Homogenous populations of U-2 OS cells were established by expanding the monoclonal populations from single U-2 OS cells. For the generation of drug-resistant subpopulations the cells were treated with 5 µM Idasanutlin for 12–20 days. The resistant populations were visualised by in situ staining of the growing cells with BrdU/FITC-anti-BrdU and Hoechst 33 342.Results and discussionsThe treatment of U-2 OS cells with Idasanutlin leads to a strong induction of p53 expression level and the expression of p53-target gene encoding p21 protein. As a result Idasanutlin induces tremendous cell cycle arrest. However no apoptosis is induced and Idasanutlin -treated U-2 OS cells can be cultured for long time periods in the presence of the drug. This prolonged treatment of U-2 OS cells leads to de novo generation of cell populations that escape from the cytostatic effect evoked by Idasanutlin.ConclusionAlthough modern Mdm2 antagonists have much improved activities compared to their precursors (i.e. nutlin-3), they suffer from similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug resistant subpopulations.

Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

Attempts at eradicating metastatic cancers with targeted therapies are limited by the emergence of resistant subclones bearing heterogeneous (epi)genetic changes. We used colorectal cancer (CRC) to test the hypothesis that interfering with an ancestral oncogenic event shared by all the malignant cells (such as WNT pathway alterations) could override heterogeneous mechanisms of acquired drug resistance. Here, we report that in CRC-resistant cell populations, phylogenetic analysis uncovers a complex subclonal architecture, indicating parallel evolution of multiple independent cellular lineages. Functional and pharmacological modulation of WNT signalling induces cell death in CRC preclinical models from patients that relapsed during the treatment, regardless of the drug type or resistance mechanisms. Concomitant blockade of WNT and MAPK signalling restrains the emergence of drug-resistant clones. Reliance upon the WNT–APC pathway is preserved throughout the branched genomic drift associated with emergence of treatment relapse, thus offering the possibility of a common therapeutic strategy to overcome secondary drug resistance.

Subtractive Genomics, Molecular Docking and Molecular Dynamics Simulation Revealed LpxC as a Potential Drug Target Against Multi-Drug Resistant Klebsiella pneumoniae

The emergence and dissemination of pan drug resistant clones of Klebsiella pneumoniae are great threat to public health. In this regard new therapeutic targets must be highlighted to pave the path for novel drug discovery and development. Subtractive proteomic pipeline brought forth UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase (LpxC), a Zn+2 dependent cytoplasmic metalloprotein and catalyze the rate limiting deacetylation step of lipid A biosynthesis pathway. Primary sequence analysis followed by 3-dimensional (3-D) structure elucidation of the protein led to the detection of K. pneumoniae LpxC (KpLpxC) topology distinct from its orthologous counterparts in other bacterial species. Molecular docking study of the protein recognized receptor antagonist compound 106, a uridine-based LpxC inhibitory compound, as a ligand best able to fit the binding pocket with a Gold Score of 67.53. Molecular dynamics simulation of docked KpLpxC revealed an alternate binding pattern of ligand in the active site. The ligand tail exhibited preferred binding to the domain I residues as opposed to the substrate binding hydrophobic channel of subdomain II, usually targeted by inhibitory compounds. Comparison with the undocked KpLpxC system demonstrated ligand induced high conformational changes in the hydrophobic channel of subdomain II in KpLpxC. Hence, ligand exerted its inhibitory potential by rendering the channel unstable for substrate binding.

Evaluation of Ceftolozane-Tazobactam in Combination with Meropenem against Pseudomonas aeruginosa Sequence Type 175 in a Hollow-Fiber Infection Model.

The aim of this study was to investigate the efficacy of ceftolozane-tazobactam in combination with meropenem against an extensively drug-resistant (XDR) Pseudomonas aeruginosa high-risk clone, sequence type 175, isolated in a Spanish university hospital. A 14-day hollow-fiber infection model was used to simulate clinical exposure of the two drug regimens alone and in combination, and serial samples were collected to determine drug concentrations and CFU counts. The untreated control failed, as did each study regimen when administered alone. However, when ceftolozane-tazobactam was administered in combination with meropenem, there was a >4-log10 CFU/ml bacterial density reduction and suppression of resistance for the duration of the study. These data suggest that ceftolozane-tazobactam plus meropenem may be a useful combination for treating XDR P. aeruginosa.