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Background and Hypothesis: Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. They are largely characterized into M1 or M2 types. TAMs express an anti-inflammatory M2-like phenotype, promote tumor progression. However, the role of M2-TAMs in driving disease pathogenesis in patients with Juvenile myelomonocytic leukemia (JMML), a rare form of pediatric leukemia driven to a large extent by mutations in the PTPN11 gene, which encodes the phosphatase SHP2 is unclear. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-TAMs express higher levels of arginase-1 (Arg-1) and PD-L1, which may contribute to the local suppression of immune responses and support the development of JMML. Methods: To study how alterations in M1/M2 macrophages contribute to JMML development, we utilized a mouse model bearing Shp2E76K mutation (Ptpn11E76K/+) which manifests the cardinal features of human JMML. We hypothesized that Shp2E76K/+ mutations enhance the function of bone marrow derived macrophages (BMDMs), including M2-TAMs and contribute to T-cell suppression. Results: Our analysis of the bulk RNA-sequence data from 90 JMML patients showed increase in the expression of Arg-1 and PD-1. Furthermore, single cell RNA-seq analysis of macrophages from 4 JMML patients revealed higher expression of M2-macrophage markers/genes. Our results show that in M2-TAMs, Arg-1 and PD-L1 levels are elevated in BM and spleens of Shp2E76K/+ mice compared to WT. Moreover, M2-TAMs, Arg-1 and PD-L1 levels were also higher in BMDMs derived from Shp2E76K/+ mice compared to WT. The BMDMs from Shp2E76K/+ mice have greater proliferation and migration potential compared to WT BMDMs, which was significantly reduced by inhibiting the function of Arg-1 and PD-L1. Conclusion: Our results show that M2-TAMs, arginase-1, and PD-L1 create a pro-tumor microenvironment, which likely contributes to the growth of JMML cells. Inhibition of Arg-1 and PD-L1 is a novel therapeutic approach to treat patients with JMML.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus, sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited Km of 11.06 ± 0.05 mmol·L−1 for glutamate and 2.4 ± 0.03 mmol·L−1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus.

Obesity is an increasing epidemic disease world-wide responsible for enhancing the risk for developing Type 2 diabetes mellitus (T2DM) as well as cancer. However, it is unclear if and how obesity contributes to the transformation of pre-leukemic stem and progenitors (pre-LHSC/Ps) into full-blown leukemia such as acute myeloid leukemia (AML) or severe form of myeloproliferative neoplasm (MPN). We hypothesized that obesity induced chronic inflammation might be responsible for clonal selection of pre-LHSC/Ps bearing pre-leukemic mutations such as DNA methyltransferase 3A (DNMT3A) and for promoting the progression of early-onset MPN towards severe forms of AML/leukemia. To test this hypothesis, we genetically crossed pre-leukemic Dnmt3a+/-;Mx-Cre+ mice with leptin deficient obese (LepOb/Ob) mice to obtain Ob/Ob;Dnmt3a+/-;Mx-Cre+ compound mutant mice. Further, the Dnmt3a gene was deleted by giving the PolyIC and the deletion was confirmed through PCR. After 12 days of post-PolyIC the myeloid cells (neutrophils and monocytes) were expanded in Ob/Ob;Dnmt3a+/-;Mx-Cre+ mice compared to Dnmt3a+/-;Mx-Cre+, Dnmt3a+/-;Mx-Cre-, Ob/Ob and WT mice. We have harvested and analyzed all these mice after 26 days of post-PolyIC. Interestingly, Ob/Ob;Dnmt3a+/-;Mx-Cre+ mice showed increased BM cellularity, both the frequency of lineage negative, Sca-1+ and c-KIT+ (LSK) cells, short-term hematopoietic stem cells (ST-HSCs; LSK/CD48+/CD150-), granulocyte macrophage progenitor (GMPs; LSK/CD16+/CD34+), and reduction in LT-HSCs (LT-HSCs; LSK/CD48-/CD150+) compared to other groups. Flow cytometry analysis of PB, BM and spleen from Ob/Ob;Dnmt3a+/-;Mx-Cre+ mice demonstrated a significant increase in the frequency of mature myeloid cells (Gr-1+/Mac-1+) and a profound reduction in B220+ B cells compared to other groups. Remarkably, these mice also showed splenomegaly, elevated heart size and early signs of AML blasts as reflected by the presence of c-KIT+/CD11b+ double positive cells in the BM, consistent with severe MPN/AML development. Taken together, these results demonstrate that obesity induced inflammation cooperates with pre-leukemic Dnmt3a+/- mutation to induce an early-onset of severe MPN/AML like disease.

Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. Macrophages are largely characterized as M1 or M2 types, and TAMs have been shown to express an M2-like phenotype. TAMs endorse tumor progression and contribute to resistance to chemotherapies. However, it is unclear what the composition of M2 macrophages is in patients with Juvenile myelomonocytic leukemia (JMML) and how do these cells mechanistically contribute to JMML and/or relapse after bone marrow transplantation. To study the role of M2- TAMs in JMML development, we first examined the bulk RNA-sequence data in 90 JMML patients. These data demonstrated a significant increase in the expression of arginase-1 (Arg-1) and programmed cell death-1 (PD-1). Furthermore, single cell RNA-sequencing analysis of monocytes/macrophages from 4 JMML patients revealed higher expression of M2- macrophage markers/genes such as IL-10, CD163, MRC1/CD206, TGF-β1 and IL-1R1 compared to M1 macrophage (CD80, CCR7, IL-6, CXCL10, CXCL11 and TNF) expression. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-macrophages express higher levels of arginase and PD-1, which may contribute to the local suppression of immune responses and damage the bone marrow microenvironment (BME) leading to poor engraftment of normal donor cells, resulting in relapse. To study how alterations in bone marrow (BM) macrophages (M1/M2) contribute to JMML development and relapse, we utilized a mouse model bearing Shp2 E76K mutation (Ptpn11 E76K/+) driven by lysosome-cre (Ptpn11 E76K/+; LysM-Cre+, indicated as Shp2* mice hereafter). This model is frequently used to study JMML as it manifests cardinal features of human JMML. In a competitive transplantation experiment using, Shp2* + Boy/J BM cells (1:1 ratio) transplanted into lethally irradiated Shp2* recipient mice, we show that Shp2* mutant cells out compete WT BoyJ cells and result in rapid growth of CD45.2+ Shp2* mutant mature myeloid cells, hematopoietic stem and progenitors (HSC/Ps) and M2- macrophages (F4/80+/CD206+) in the BM and spleen leading to leukemia relapse. To determine if modulating Arg-1 and PD-1/PD-L1 levels in the background of Shp2* mutant leukemic stem cells in Shp2* recipients would alter the overall engraftment and JMML development and relapse, we again performed a competitive transplantation experiment using, Shp2* + Boy/J (BM cells, 1:1 ratio) into Shp2* and WT recipient mice. After 8 weeks post transplantation, we investigated the role of Arg-1 and PD-L1 in Shp2* recipients using pharmacological inhibitors, CB-1158 (Arg-1 inhibitor; 100 mg/kg, orally) + anti-PD-L1 antibody (10 mg/kg, i.p) for 30 days. The Arg-1 + PD-L1 treatment significantly reduced the number of white blood cells, neutrophils, monocytes and improved RBC and platelet counts. The spleen and liver weights were significantly rescued as well. Interestingly, CD45.1 WT donor cells in the PB, BM, and spleen were significantly increased and a significant reduction of Shp2* mutant CD45.2+ mature myeloid cells in the PB, BM, and spleen was observed. Importantly, the frequency and absolute number of leukemic blasts, LSK (Lin-/Sca1+/c-KIT+) cells, short term hematopoietic cells (ST-HSCs), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP) were significantly reduced. Furthermore, the M2- TAMs were significantly reduced in the BM and spleen of Arg-1 + PD-L1 drug treated group compared to vehicle treated mice. Notably the CD8+ T-cells (IFN-γ+ and TNF-α+) were significantly improved in the drug treated mice. These data suggest that the suppression of arginase-1 allows for the arginine levels to increase, which promotes the proliferation of T-cells. Increasing arginine levels also promotes an anti-tumor immune response resulting in the emergence of CD45.1 WT HSCs as opposed to mutant CD45.2 HSCs, suggesting that Arg-1 + PD-L1 treatment is a novel therapeutic approach to treat patients with JMML and for preventing leukemia relapse after BM transplantation. Disclosures No relevant conflicts of interest to declare.

Obesity is an increasing epidemic world-wide responsible for enhancing the risk for developing Type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and cancer. However, it is unclear if and how obesity contributes to the transformation of pre-leukemic stem and progenitors (pre-LHSC/Ps) into full-blown leukemia such as acute myeloid leukemia (AML) or severe form of myeloproliferative neoplasm (MPN) or CVD. We hypothesized that obesity induced chronic inflammation might be responsible for clonal selection of pre-LHSC/Ps bearing pre-leukemic clonal hematopoiesis of indeterminate potential (CHIP) mutations such as DNMT3A, TET2, ASXL1, and JAK2 and for promoting the progression of early-onset MPN, AML/leukemia and CVD. To study the linkage between obesity and CHIP in humans, we first examined the UK biobank. After exclusions, the final study cohort included 47,466 unrelated participants free of T2DM at baseline and having valid CHIP measurements. The mean (SD) age at enrollment was 56.5 (8.0), 45.0% were male, 43.9% never smoked, and 82.6% self-reported as European decedents. At baseline, the mean (SD) body mass index (BMI) was 27.3 (4.7) kg/m 2, with 43.0% overweight and 23.6% obese, and the overall mean (SD) waist-to-hip ratio (WHR) was 0.87 (0.09). CHIP was present among 5.8% of the study population the most common mutations on the DNMT3A (3.7%) and TET2 (1.0%) genes; large CHIP clone defined as CHIP mutation with variant allele fraction >10% was present among 2.4% of the study population. Individuals with CHIP mutations on average had higher WHR. The presence of CHIP mutation was associated with a 0.0028 increase of WHR (p=0.03). Furthermore, CHIP prevalence increased with higher WHR: the percentage of participants with CHIP was 4.93%, 5.75%, 6.56% in the lowest, middle, and highest WHR quintiles respectively, signifying that dysfunctional metabolism may accelerate expansion of clonal hematopoiesis (CH). To better define the mechanism of obesity driven CH, we utilized several novel mouse models bearing Tet2 -/-, Dnmt3a +/-, Asxl1 +/- and Jak2 +/- mutations to mimic the human pre-LHSC/Ps condition and obesity, in the form of leptin deficient Lep Ob/Ob (Ob/Ob) mutation, which induces obesity and T2DM. We show that both the compound mutant (Tet2 -/-;Ob/Ob, Dnmt3a +/-;Ob/Ob, Asxl1 +/-;Ob/Ob and Jak2 +/-;Ob/Ob) and CHIP mutant bone marrow (BM; Tet2 -/-, Dnmt3a +/-, Asxl1 +/- and Jak2 +/-) transplanted into Ob/Ob mice develop rapid growth of mature myeloid cells and HSC/Ps leading to severe form of MPN/AML as well as CVD. This was associated with upregulation of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. Flow cytometry analysis of LSK and progenitor cells isolated from Tet2 -/-;Ob/Ob mice revealed an up-regulation of intracellular Ca2+ levels. We hypothesized that up-regulated Ca2+ signaling in Tet2 -/-; Ob/Ob HSC/Ps promotes aberrant signaling leading to an early-onset of severe MPN/AML. We performed a competitive transplantation experiment using, Tet2 -/-: Boy/J BM cells (1:1 ratio) into Ob/Ob and WT recipients. After 8 weeks post transplantation, we investigated the role of Ca2+ blockers in driving CH in Ob/Ob recipients using pharmacological inhibitors, either individually, or in combination, of metformin (100 mg/kg, orally), nifedipine (100 mg/kg, orally), MCC950 (30 mg/kg, orally) and anakinra (10 mg/kg, i.p). The combination treatment markedly reduced monocytes, neutrophils, WBC counts, and improved RBCs, hematocrits and platelets. The spleen and liver, heart, body weights and blood glucose levels were significantly reduced, along with a greater re-emergence of normal CD45.1 wild-type cells in the PB, BM, and spleen and a significant reduction in Tet2 mutant CD45.2 pre-LHSC/Ps and myeloid cells in the PB, BM, and spleen. Importantly, the frequency of leukemic blasts, LSK cells, ST-HSCs, LT-HSCs and granulocyte macrophage progenitors (GMP) were significantly reduced. Furthermore, the combination of drug treatment showed greater heart protective activity by reducing the atherosclerotic lesions in Ob/Ob recipients bearing CHIP by suppressing Ca2+ signaling. Taken together, these data suggest that obesity is highly associated with the presence of CHIP in humans and that targeting CHIP mutant cells with a combination of metformin/nifedipine/MCC950/anakinra is a safe and inexpensive way to rescue CH and its associated leukemic and cardiovascular defects. Disclosures Natarajan: Amgen: Research Funding; Apple: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Boston Scientific: Research Funding; Blackstone Life Sciences: Consultancy; Genentech: Consultancy; Foresite Labs: Consultancy.

Congenital erythrocytosis (CE) can be classified as primary and secondary and 82 consecutive patients of erythrocytosis who were JAK-2 mutation negative, were further investigated. The genomic DNA was extracted from all the patients and the EPO-R, VHL, EGLN1 and EPAS1 genes were PCR amplified and sequenced. The sequence analysis showed (28/82) 34.14% patients had mutations. Among them, (19/28) 67.86% patients had mutations in exon 8 of EPO-R gene, of which six were novel missense mutations, p.(Gly418Ala), p.(Gly390Ala), p.(Ala411Thr), p.(Gly475Val), p.(Glu490Asp), p.(Glu362Gln) and three were novel frameshift mutations, p.(Glu336*), p.(Pro327Hisfs*68), p.(Gly479Alafs*37). All these EPO-R patients were heterozygotes and were forming endogenous erythrocyte colonies (EEC). Some patients (8/28) 28.57% had mutations in VHL gene, out of which 3 novel homozygous missense mutations in exon 1 of VHL gene, p.Gly80Asp, p.Gln107Glu and p.Gln113Glu, were identified. In addition, (1/28) 3.5% patients had one reported heterozygous missense mutation in exon 12 of EPAS1 gene p.Gly537Arg and one novel frameshift mutation p.(Ala553Glyfs*58). Further, in silico analysis indicated most of the mutations, probably, were damaging the protein structures, causing the CE in these patients. In this study the mutations in EPO-R and EPAS1 genes were identified for the first time in India.

In Staphylococcus aureus 85% of glucose is catabolised through EMP pathway and the very first step of the formation of glucose-6-phosphate (G-6-P) in the cytoplasm is catalysed by glucokinase (glck) which was isolated and purified from S. aureus ATCC12600. This G-6-P formation has essential role in the pathogen from energy generation in the catabolic reactions to the synthesis of all the intermediates for the very survival of S. aureus. This enzyme was purified by 20–40% ammonium sulphate fractionation and Diethylaminoethyl (DEAE) cellulose ion exchange chromatography followed by reverse phase high performance liquid chromatography (RP-HPLC) the glck was eluted at a retention time of 15 min. The purified glck in 10% SDS-PAGE showed single band with a molecular weight of 33 kDa confirming the monomeric in nature. The pure glck obtained from DEAE cellulose ion exchange chromatography followed by RP-HPLC exhibited Km of 5.22 ± 0.17 mM and Vmax 2.24 ± 0.06 with Hill coefficient of 1.71 ± 0.025 mM. The enzyme kinetics showed very high degree of cooperativity towards glucose which means this enzyme is highly involved in the phosphorylation glucose in S. aureus.

Wilms' tumor-suppressor gene-1 (WT1) is a transcription factor that contains four zinc-finger motifs at the C-terminus and plays a crucial role in kidney and gonad development. We have identified primitive glomeruloid formation using immunohistochemistry in a patient who was clinically diagnosed with a Wilms' tumor. In order to understand the involvement of mutations in the WT1 gene, the genomic DNA was isolated from peripheral blood of the patient (18/F). Exon 9 of the WT1 gene was amplified and sequenced. The obtained sequence was BLAST searched against the transcript variants (TV) of the WT1 gene. An amplified exon 9 sequence of the WT1 gene showing similarity with exon 9 of TV-A, F and exon 10 of TV-B, D and E with a deletion of single nucleotide 'A' causing frame shift in the 4th zinc finger domain of the WT1 protein resulted in Wilms' tumor condition. The deletion position is variable with different transcript variants and they are present at: for TV-A c.1592delA, p.468, for TV-F c.1053delA, p.259, for TV-B c.1643delA, p.485, for TV-D c.1652 delA, p.488, and for TV-E c.1095delA, p.273; all these variations resulted in frame shift mutation. In order to substantiate these results in silico analysis was carried out; the structural superimposition of wild type and mutant WT1 structures showed that the mutated region exhibited a different confirmation with RMSD of 1.759A. Therefore, these results conclusively explain the mutation in the WT1 gene that leads to structural changes contributing to glomerulosclerosis.

Glucokinase (GK) plays a critical role in glucose homeostasis and the mutations in GK gene result in pathogenic complications known as Maturity Onset Diabetes of the Young 2, an autosomal dominant form of diabetic condition. In the present study, GK was purified from human liver tissue and the pure enzyme showed single band in SDS-PAGE with a molecular weight of 50 kDa. The kinetics of pure GK showed enzyme activity of 0.423±0.02 µM glucose-6-phosphate (G6P)/mL/Min and Km value of 6.66±0.02 µM. These values were compared in the liver biopsy of a clinically proven type 2 diabetic patient, where GK kinetics showed decreased enzyme activity of 0.16±0.025 µM G6P/mL/Min and increased Km of 23±0.9 µM, indicating the hyperglycemic condition in the patient. The genetic analysis of 10th exon of GK gene from this patient showed a R308K mutation. To substantiate these results, comparative molecular dynamics and docking studies were carried out where a higher docking score (-10.218 kcal/mol) was observed in the mutated GK than wild-type GK structure (-12.593 kcal/mol) indicating affinity variations for glucose. During the simulation process, glucose was expelled out from the mutant conformation but not from wild-type GK, making glucose unavailable for phosphorylation. Therefore, these results conclusively explain hyperglycemic condition in this patient.