Analysis of 5-Azacytidine Resistance Models Reveals a Set of Targetable Pathways

The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of mechanisms by which the MDS cell becomes to tolerate HMA and progresses to acute myeloid leukemia (AML) requires the development of new cellular models. From MDS/AML cell lines we developed a model of 5-azacytidine (AZA) resistance whose stability was validated by a transplantation approach into immunocompromised mice. When investigating mRNA expression and DNA variants of the AZA resistant phenotype we observed deregulation of several cancer-related pathways including the phosphatidylinosito-3 kinase signaling. We have further shown that these pathways can be modulated by specific inhibitors that, while blocking the proliferation of AZA resistant cells, are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms that can be targeted to expand therapeutic options during progression on AZA therapy.

Sensory Afferent Renal Nerve Activated Gαi2 Subunit Proteins Mediate the Natriuretic, Sympathoinhibitory and Normotensive Responses to Peripheral Sodium Challenges

We have previously reported that brain Gαi2 subunit proteins are required to maintain sodium homeostasis and are endogenously upregulated in the hypothalamic paraventricular nucleus (PVN) in response to increased dietary salt intake to maintain a salt resistant phenotype in rats. However, the origin of the signal that drives the endogenous activation and up-regulation of PVN Gαi2 subunit protein signal transduction pathways is unknown. By central oligodeoxynucleotide (ODN) administration we show that the pressor responses to central acute administration and central infusion of sodium chloride occur independently of brain Gαi2 protein pathways. In response to an acute volume expansion, we demonstrate, via the use of selective afferent renal denervation (ADNX) and anteroventral third ventricle (AV3V) lesions, that the sensory afferent renal nerves, but not the sodium sensitive AV3V region, are mechanistically involved in Gαi2 protein mediated natriuresis to an acute volume expansion [peak natriuresis (μeq/min) sham AV3V: 43 ± 4 vs. AV3V 45 ± 4 vs. AV3V + Gαi2 ODN 25 ± 4, p < 0.05; sham ADNX: 43 ± 4 vs. ADNX 23 ± 6, AV3V + Gαi2 ODN 25 ± 3, p < 0.05]. Furthermore, in response to chronically elevated dietary sodium intake, endogenous up-regulation of PVN specific Gαi2 proteins does not involve the AV3V region and is mediated by the sensory afferent renal nerves to counter the development of the salt sensitivity of blood pressure (MAP [mmHg] 4% NaCl; Sham ADNX 124 ± 4 vs. ADNX 145 ± 4, p < 0.05; Sham AV3V 125 ± 4 vs. AV3V 121 ± 5). Additionally, the development of the salt sensitivity of blood pressure following central ODN-mediated Gαi2 protein down-regulation occurs independently of the actions of the brain angiotensin II type 1 receptor. Collectively, our data suggest that in response to alterations in whole body sodium the peripheral sensory afferent renal nerves, but not the central AV3V sodium sensitive region, evoke the up-regulation and activation of PVN Gαi2 protein gated pathways to maintain a salt resistant phenotype. As such, both the sensory afferent renal nerves and PVN Gαi2 protein gated pathways, represent potential targets for the treatment of the salt sensitivity of blood pressure.

Resistance profiling of Aspergillus fumigatus to olorofim indicates absence of intrinsic resistance and unveils the molecular mechanisms of acquired olorofim resistance

Olorofim (F901318) is a new antifungal currently under clinical development that shows both in vitro and in vivo activity against a number of filamentous fungi including Aspergillus fumigatus. In this study we screened A. fumigatus isolates for intrinsic olorofim-resistant A. fumigatus and evaluated the ability of A. fumigatus to acquire an olorofim-resistant phenotype. No intrinsic resistance was found in 975 clinical A. fumigatus isolates. However, we found that isolates with increased olorofim MICs (> 8 mg/L) could be selected using a high number of conidia and olorofim exposure under laboratory conditions. Assessment of the frequency of acquired olorofim resistance development of A. fumigatus was shown to be higher than for voriconazole but lower than for itraconazole. Sequencing the PyrE gene of isogenic isolates with olorofim MICs of >8 mg/L identified various amino acid substitutions with a hotspot at locus G119. Olorofim was shown to have reduced affinity to mutated target protein dihydroorotate dehydrogenase (DHODH) and the effect of these mutations were proven by introducing the mutations directly in A. fumigatus. We then investigated whether G119 mutations were associated with a fitness cost in A. fumigatus. These experiments showed a small but significant reduction in growth rate for strains with a G119V substitution, while strains with a G119C substitution did not exhibit a reduction in growth rate. These in vitro findings were confirmed in an in vivo pathogenicity model.

1230. Evaluation of the Interplay Between β-lactamases and Porin Production on β-Lactam MICs in Klebsiella pneumoniae

Background K. pneumoniae can emerge resistant to β-lactam antibiotics through the production of β-lactamase enzymes and/or loss of the outer membrane porins, OmpK35, OmpK36, and/or PhoE. While both mechanisms are hypothesized to work synergistically, β-lactamases have been the focus of previous studies. As a result, the contribution of outer membrane porin loss to the β-lactam minimum inhibitory concentration (MIC) is unknown. The objective of this study was to evaluate the contribution of specific β-lactamases and porin production to β-lactam susceptibility. We hypothesize that production of a β-lactamase in a clinical isolate deficient in 3 major porins will result in higher β-lactam MICs but not always a resistant phenotype. Methods The structural gene and promoter of CTX-M-14, CTX-M-15, and CMY-2 were cloned into a low copy number vector and transformed into Kp 23, a wild-type clinical isolate, and KPM 20, a clinical isolate deficient in OmpK35/36 and PhoE. MICs to ceftolozane/tazobactam, cefotaxime, ceftazidime, cefepime, and meropenem were determined by E-test. Kp 23 and KPM 20 were characterized by Western blot and whole genome sequencing. Results Production of CMY-2 alone led to a resistant phenotype for ceftolozane/tazobactam, cefotaxime, and ceftazidime regardless of porin production (Figure 1). CMY-2 production in KPM 20 resulted in non-susceptibility to meropenem. Both clones were susceptible to cefepime. Production of CTX-M-14 and CTX-M-15 in Kp 23 resulted in only cefotaxime resistance. Production of CTX-M-14 and CTX-M-15 in KPM 20 resulted in isolates non-susceptible to all antibiotics tested. Figure 1. MICs of K. pneumoniae clones against panel of β-lactam antibiotics. Conclusion When evaluating clinical isolates, it is impossible to determine the contribution of individual resistance mechanisms in the susceptibility pattern. This study demonstrated that resistance is not solely dependent on the β-lactamase produced and that the impact of porin deficiency varies with the antibiotic being evaluated. These data suggest that antibiotic selection may be more nuanced and that a broader range of therapeutics may be available given the appropriate diagnostic tools. Understanding the contributions of all resistance mechanisms is necessary to inform selection of the most appropriate antibiotic therapy. Disclosures Nancy D. Hanson, PhD, Merck (Grant/Research Support)

Marker-Assisted Evaluation of Two Powdery Mildew Resistance Candidate Genes in Korean Cucumber Inbred Lines

Two genes, CsLRR-RPK2 (CsGy5G015660) and CsaMLO8 (Csa5G623470), have been considered as powdery mildew (PM) resistance genes in cucumbers. In this study, we evaluated the involvement of the alleles of these two genes in PM resistance in 100 commercial Korean cucumber inbred lines. To achieve this, we developed cleaved amplified polymorphic sequences (CAPS) and InDel markers from CsLRR-RPK2 and CsaMLO8. Genotyping analysis indicated that the CsLRR-RPK2-CAPS marker showed a stronger correlation with the PM-resistant phenotype, with an 84% consistency compared to the CsaMLO8-InDel marker. The use of the CsaMLO8-InDel marker showed a 70% consistency between phenotype and genotype results. It was proposed that the CsLRR-RPK2-CAPS marker successfully eliminated PM-susceptible inbred lines, since both genotype and phenotype results were 100% identical. Furthermore, the present study revealed that the introduction of one of these alleles is probably enough to confer PM resistance in cucumbers. However, seven PM-resistant inbred lines harbored either CsaMLO8 or CsLRR-RPK2 alleles, indicating that there is another PM-resistant resource(s) besides CsaMLO8- and CsLRR-RPK2–originated resistance in the commercial Korean inbred lines. Our results provide reliable evidence confirming two PM-resistant candidate genes for the detection of PM resistance resources in cucumber inbred lines.

Menin inhibition suppresses castration-resistant prostate cancer and enhances chemosensitivity

Disease progression and therapeutic resistance of prostate cancer (PC) are linked to multiple molecular events that promote survival and plasticity. We previously showed that heat shock protein 27 (HSP27) acted as a driver of castration-resistant phenotype (CRPC) and developed an oligonucleotides antisense (ASO) against HSP27 with evidence of anti-cancer activity in men with CRPC. Here, we show that the tumor suppressor Menin (MEN1) is highly regulated by HSP27. Menin is overexpressed in high-grade PC and CRPC. High MEN1 mRNA expression is associated with decreased biochemical relapse-free and overall survival. Silencing Menin with ASO technology inhibits CRPC cell proliferation, tumor growth, and restores chemotherapeutic sensitivity. ChIP-seq analysis revealed differential DNA binding sites of Menin in various prostatic cells, suggesting a switch from tumor suppressor to oncogenic functions in CRPC. These data support the evaluation of ASO against Menin for CRPC.

Phenotypic and Genotypic Characterization of Salmonella spp. Isolated from Foods Involved in Human Salmonellosis Outbreaks in Minas Gerais State, Brazil

Salmonella spp. is one of the primary pathogens that cause foodborne diseases worldwide. In the present study, we deeply characterized Salmonella spp. originated from foods related to human salmonellosis outbreaks in Minas Gerais – Brazil, from 2003 to 2017. In this regard, the serotyping, antimicrobial susceptibility, virulence genes and genetic polymorphism determined by rep-PCR were performed in 70 Salmonella spp. isolates. Thirteen serotypes of Salmonella spp. were identified, and S. Enteritidis and S. Typhimurium were the most prevalent, both corresponding to 74.3% (52/70) of all isolates. Sixty-five (92.8%) isolates demonstrated to be resistant to at least one of the 15 antimicrobials tested. Ten isolates (14.2%) showed a multidrug-resistant phenotype. Sixteen virulence genes were screened and detection ranged from 75.7 to 100% of all isolates. There was a statistical difference among Salmonella spp. serotypes in detecting the sipB , sopE , lfpA , sefA and spvC genes. Forty isolates of S. Enteritidis from 16 outbreaks were grouped into 14 fingerprints, while 12 S. Typhimurium retrieved six fingerprints. The bacteria presented a serological pattern like those reported by main public health centers worldwide. One major concern of our findings is the high levels of both detection of virulence genes and resistance to antimicrobials, especially to critically important drugs. In this concern, especial attention should be given to the serotype S. Enteritidis. Although the isolates of Salmonella spp. have presented a relatively variable genome, high genetic similarity was observed among them, with some of them sharing identical fingerprints. These results corroborate the hypothesis of clonal circulation of Salmonella spp. in human infections in Minas Gerais.

Diversity and Resistance Profiles of Human Non-typhoidal Salmonella spp. in Greece, 2003–2020

Salmonella spp. is one of the most common foodborne pathogens in humans. Here, we summarize the laboratory surveillance data of human non-typhoidal salmonellosis in Greece for 2003–2020. The total number of samples declined over the study period (p < 0.001). Of the 193 identified serotypes, S. Enteritidis was the most common (52.8%), followed by S. Typhimurium (11.5%), monophasic S. Typhimurium 1,4,[5],12:i:- (4.4%), S. Bovismorbificans (3.4%) and S. Oranienburg (2.4%). The isolation rate of S. Enteritidis declined (p < 0.001), followed by an increase of the less common serotypes. Monophasic S. Typhimurium has been among the five most frequently identified serotypes every year since it was first identified in 2007. Overall, Salmonella isolates were resistant to penicillins (11%); aminoglycosides (15%); tetracyclines (12%); miscellaneous agents (sulphonamides, trimethoprim, chloramphenicol and streptomycin) (12%) and third-generation cephalosporins (2%). No isolate was resistant to carbapenems. In total, 2070 isolates (24%) were resistant to one or two antimicrobial classes and 903 (10%) to three and more. Out of the 1166 isolates resistant to fluoroquinolones (13%), 845 (72%) were S. Enteritidis. S. Enteritidis was also the most frequently identified serotype with a resistance to third-generation cephalosporins (37%, 62/166), followed by S. Typhimurium (12%, 20/166). MDR was most frequently identified for S. Typhimurium and its monophasic variant (resistant phenotype of ampicillin, streptomycin, tetracycline and sulphamethoxazole with or without chloramphenicol or trimethoprim).