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Tumors emerge and evolve in complex and dynamic microenvironments that influence their growth, invasion, and metastasis. In the tumor microenvironment, the immune system plays a critical role in the prevention and elimination of tumors. Despite immune surveillance, tumors still manage to develop through evolution facilitating the establishment of an immune suppressive microenvironment. There is emerging evidence showing that the antitumor immunity of immune effector cells can be obstructed by immune suppressive cell populations such as MDSCs or Tregs. Hematologic malignancies associated with FGFR1 translocations, also known as Stem Cell Leukemia/Lymphoma (SCLL) syndrome, show a complex presentation including myeloproliferative neoplasm, AML, and T- or B-cell lineage lymphoblastic leukemia/lymphoma, providing an ideal, homogeneous model to investigate mechanisms underlying leukemogenesis. Our recent investigation in mouse models of SCLL revealed that mice xenografted with SCLL cells showed increased CD11b+Ly6CHigh M-MDSC and CD11b+Ly6CInt PMN-MDSC levels in the peripheral blood (PB) during leukemia development, accompanied by significantly decreased levels of T cells. To further confirm this MDSC-mediated suppression of T cell function, MDSC depletion experiments were performed in the SCLL cell engraftment model using either the anti-Ly6G antibody (for PMN-MDSCs) or the anti-Gr-1 (aka Ly6G/Ly6C; for total MDSCs) antibody. In both cases, significantly increased levels of CD4+ and CD8+ cells were observed, along with significantly prolonged mouse survival. These observations were consistent in multiple SCLL cell line models, supporting the idea that SCLL cells promote the accumulation of MDSCs, which in turn suppresses the antitumor activity of cytotoxic T cells, allowing SCLL progression. To further characterize these leukemia cell induced MDSCs, CyTOF analysis was performed with the PBMC and bone marrow (BM) samples from leukemic and naïve mice. In peripheral blood leukemia cell induced M-MDSCs also express macrophage markers, including F4-80 and CD64, implying a potential overlap of MDSC and tumor-associated macrophage (TAM). These TAMs also express high CD71 and CD184. Most importantly, we can also identify a CD64/CD71/CD184 expressing cluster in the BM samples. Different from the PB, these cells in the BM express stem cell marker CD117, suggesting there are potential progenitor cells of TAMs. Taken together, our studies demonstrated that SCLL cells establish a tumor favorable microenvironment by reshaping hematopoiesis toward MDSC generation in the BM. A comprehensive understanding of these immune modulation processes holds the promise of improving patient outcomes by reversing the generation of MDSC/TAM in the BM and overturning the establishment of the immunosuppressive microenvironment at the outset. Citation Format: Xiaocui Lu, Ting Zhang, Atsuko Matsunaga, Xuexiu Fang, Stephanie F. Mori, John Cowell, Tianxiang Hu. CD71 and CD117 identifies an early cancer associated macrophage progenitor population in leukemic mouse bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1353.

Hematologic malignancy associated with fibroblast growth factor receptor 1 (FGFR1) translocations develops an aggressive stem cell leukemia/lymphoma (SCLL), 80% of which progress to AML and only ~15% show survival of > 15 months. In addition, 23.6% of de novo AML patients overexpress FGFR1, compared with healthy individuals. Therefore, investigating how tumor-intrinsic FGFR1 signaling modulates the tumor microenvironment and their contribution to leukemogenesis is not only important for understanding leukemia initiation and progression but also critical for the development of an efficient cure for AML patients. Interleukin Receptor Associated Kinase 1 (IRAK1) is a protein kinase that initiates a cascade of signaling events eventually leading to activation of proinflammatory genes, including IL-6, IL-10, TNF-α, and IFN-γ, which play critical roles in inflammation and innate immunity. The role of IRAK1 has been reported in several different subtypes of leukemia, including B cell-like diffuse large B-cell lymphoma (ABC DLBCL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and melanoma. Most previous IRAK1 studies, however, only focused on its survival and proliferation promoting roles inside the tumor cell itself. Since most of these studies engrafted human cancer cell lines into NSG mice, which lack a functional immune system, it was impossible to investigate the function of IRAK1 in modulating the tumor microenvironment. Using our well-established syngeneic SCLL mouse model, we defined IRAK1 gene as a critical target of FGFR1 in driving leukemogenesis. Our preliminary studies revealed that IRAK1 knockout (KO) in SCLL leukemia cells leads to T cell mediated clearance of the leukemia cells. In contrast, IRAK1 expressing mock control (MC) cells induce the accumulation of myeloid derived suppressor cells (MDSCs), which sequentially suppress T cell function to promote leukemia progression. These observations indicate a critical function for IRAK1 in modulating the tumor microenvironment and promoting leukemogenesis. Most importantly, pilot cytokine profiling of the MC and IRAK1 KO leukemia cells identified IFN-γ as one of the direct targets of IRAK1 in leukemia cells, and scRNA-Seq analysis revealed predominant expression of IFNGR1 and identified activation of the IFN-γ signaling pathway in leukemia induced MDSCs. Genes in this pathway include IFN-γ directly regulated genes, such as CD274 and SOCS3. Further engraftment using either IFNGR1-/- mice or IFNG KO leukemia cells revealed the involvement of this leukemia cell intrinsic, IRAK1 regulated IFN-γ in modulating the tumor microenvironment to promote leukemogenesis. In summary, our data suggests that leukemia cell-derived, IRAK1 regulated inflammatory signals act as critical modulators in establishing a tumor favorable microenvironment during leukemogenesis. Citation Format: Ting Zhang, Xiaocui Lu, Atsuko Matsunaga, Xuexiu Fang, Stephanie Fay Mori, John Cowell, Tianxiang Hu. IRAK1 regulated inflammatory signal from the leukemia cells modulates tumor microenvironment and promote leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 657.

Background Acute rejection of a kidney allograft results from adaptive immune responses and marked inflammation. The eicosanoid prostaglandin E2 (PGE2) modulates the inflammatory response, is generated by cyclooxygenase 2 (COX-2), and binds to 1 of the 4 G protein-coupled E prostanoid cell surface receptors (EP1-4). Receptor activation results in in proinflammatory (EP1 and EP3) or anti-inflammatory (EP2 and EP4) responses. We theorized that expression of the components of the COX-PGE2-EP signaling pathway correlates with acute rejection in a porcine model of allogeneic renal transplantation. Method COX-2 enzyme and EP receptor protein expression were quantitated with western blotting and immunohistochemistry from allotransplants (n = 18) and autotransplants (n = 5). Linear regression analysis was used to correlate EP receptor expression with the Banff category of rejection. Results Pigs with advanced rejection demonstrated significant increases in serum PGE2 metabolites, while pigs with less rejection demonstrated higher tissue concentrations of PGE2 metabolites. A significant negative correlation between COX-2 expression and Banff category of rejection (R = −0.877) was shown. Rejection decreased expression of EP2 and EP4. For both receptors, there was a significant negative correlation with the extent of rejection (R = −0.760 and R = −0.891 for EP2 and EP4, respectively). Rejection had no effect on the proinflammatory receptors EP1 and EP3. Conclusion Downregulation of COX-2 and the anti-inflammatory EP2 and EP4 receptors is associated with acute rejection in unmatched pig kidney transplants, suggesting that the COX-2-PGE2-EP pathway may modulate inflammation in this model. Enhancing EP2 and/or EP4 activity may offer novel therapeutic approaches to controlling the inflammation of acute allograft rejection.

Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. Stem cell transduction and transplantation in syngeneic mice were used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and the results showed that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. RPPA and RNA-Seq examination of the related signaling pathways and target genes revealed that tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expressed tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo suggesting an alternative approach for treatment of SCLL. This study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases results with the combined effects of direct protein activation by the full-length kinases and gene regulation by the truncated nucleus derivative tnFGFR1, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile. Citation Format: Baohuan Cai, Yun Liu, Yating Chong, Hualei Zhang, Stephanie Mori, Atsuko Matsunaga, Xuexiu Fang, John Cowell, Tianxiang Hu. A truncated nucleus-located derivative of FGFR1 contributes to the transformation of leukemia stem cell in leukemogenesis driven by FGFR1 fusion kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5837.

Background: VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. Copper (Cu) is essential micronutrient and has been implicated in angiogenesis. The P-type ATPase transporter ATP7A is key regulator of Cu homeostasis but its role in VEGFR2 signaling in ECs and post-ischemic neovascularization is entirely unknown. Results: Here we show that ATP7A expression was dramatically increased in the angiogenic ECs in mice hindlimb ischemia model. EC-specific ATP7A deficient mice or Cu transporter-dysfunctional ATP7A mut mice showed significant decrease in blood flow recovery and CD31+ capillary density (angiogenesis) in ischemic tissues compared to their control mice. In cultured human ECs, ATP7A knockdown with siRNA significantly inhibited VEGF-induced migration (67%), capillary network formation on Matrigel (46%). Immunofluorescence, co-immunoprecipitation, and proximity ligation assays showed that VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2. Surprisingly, loss of ATP7A promoted VEGF-induced VEGFR2 protein degradation (56%) and inhibited VEGFR2 signaling via enhancing VEGFR2 ubiquitination (2.0-fold) in a Cu-independent manner. This was associated with reduced cell surface VEGFR2 expression, increased VEGFR2 binding to selective autophagic cargo/adaptor p62/SQSTM1 (60%) and LC3-GFP+autophagic puncta (34%) and autolysosome formation (transmission electron microscopy). Inhibition of autophagy by bafilomycin A1 or chloroquine prevented enhanced VEGFR2 degradation in ATP7A-depleted ECs. Furthermore, overexpression of p62, but not p62 lacking ubiquitin binding domain, or autophagy inducer rapamycin treatment increased VEGFR2 degradation (26% and 87%, respectively) and p62 overexpression inhibited ECs migration (29%). Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by employing autophagy reporter CAG-ATP7A mut -RFP-EGFP-LC3 transgenic mice. Conclusion: Our study uncovers the unexpected and novel function of ATP7A to limit autophagic degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores neovascularization in ischemic disease.

Background: Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) have detrimental effects. Exercise not only improves endothelial dysfunction and angiogenesis in T2DM but also induces secretion of exosomes into circulation. Extracellular superoxide dismutase (ecSOD) is a major secretory Cu containing antioxidant enzyme that catalyzes dismutation of O 2 •- to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ecSOD-derived H 2 O 2 in endothelial cells (ECs) enhances angiogenesis while impaired ATP7A-ecSOD axis in diabetes induces endothelial dysfunction. Here we examined whether exercise-derived exosomes (Exe-Exo) may have pro-angiogenic effects via regulating ATP7A-ecSOD axis in T2DM. Results: Two weeks of voluntary wheel exercise of control C57Bl6 mice increased plasma exosome levels (6.2-fold) characterized by Nanosight, TEM and exosome markers (CD63, CD81, Tsg101). Treatment of HUVECs with equal number of exosomes revealed that angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold) were significantly enhanced by Exe-Exo compared to sedentary-derived exosomes (Sed-Exo). This was associated with increased ATP7A (2.9-fold) and ecSOD (1.4-fold) expression in Exe-Exo. Sed-Exo from high fat-induced T2DM mice significantly decreased EC migration (40%) and tube formation (10%) as well as ATP7A expression (28%) compared to Sed-Exo from control mice, which were restored by T2DM Exe-Exo, but not by T2DM/ecSOD KO Exe-Exo. Furthermore, exosomes overexpressing ecSOD (ecSOD-Exo) which mimic exercise increased angiogenesis and H2O2 levels in ECs, which were inhibited by overexpression of catalase. In vivo, skin wound healing model showed that direct application of T2DM Sed-Exo delayed while T2DM Exe-Exo enhanced wound healing of control mice. Furthermore, defective wound healing in T2DM mice or ecSOD KO mice were rescued by ecSOD-Exo application. Conclusion: Exercise training improves pro-angiogenic function of circulating exosomes in T2DM via increasing ATP7A-ecSOD axis, which may provide an effective therapy for promoting angiogenesis and wound repair in metabolic and cardiovascular diseases.

Background: Exercise training promotes vascular adaptation (restoration of endothelial function and angiogenesis) in type2 diabetes (T2D). Since eNOS uncoupling/O 2 - are increased in T2D, exercise may promote vascular adaptation via mechanism other than eNOS-NO axis. Extracellular superoxide dismutase (ecSOD) is a secreted copper (Cu) containing SOD that catalyzes the dismutation of O 2 - to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ATP7A-ecSOD pathway is reduced in T2D and that ecSOD-derived H 2 O 2 promotes VEGFR2 signaling and angiogenesis in endothelial cells. However, role of ATP7A-ecSOD axis and H 2 O 2 signaling in vascular adaptation to exercise in T2D have not been reported. Oxidation of Cysteine residues of targets proteins to generate cysteine sulfenic acid (Cys-OH) is a key initial event in H 2 O 2 -mediated signaling. Results: Here we show that ATP7A protein (49%), ecSOD activity (51%) and extracellular H 2 O 2 levels in blood vessels and skeletal muscles were significantly decreased in high fat diet-induced T2D mice compared to control C57Bl6 mice, which were rescued by volunteer wheel exercise (2 weeks) or in T2D/ATP7A overexpressing mice. In parallel, exercise training restored impaired endothelium-dependent relaxation (EDR) of resistant arteries and angiogenesis (CD31+ capillary, 2.4-fold) in skeletal muscle of T2D mice, which were inhibited by exogenous catalase or in T2D/ecSOD KO mice, but not by L-NAME, suggesting that ecSOD-derived H 2 O 2 , but not eNOS/NO, mediates exercise-induced beneficial effects. Mechanistically, exercise significantly increased Cys-OH formation of PKG1α (2.3-fold), which was shown to induce EDR in H 2 O 2 -, but not NO/cGMP-, dependent manner, in T2D vascular tissues as well as Cys-OH formation of AMPK (3.6-fold) and AMPK downstream angiogenic PGC1α and VEGF protein expression in T2D skeletal muscles. Of note, these exercise-induced Cys oxidation of PKG1α and AMPK were not observed in control or T2D/ecSOD KO mice. Conclusion: Exercise-induced ATP7A-ecSOD axis-mediated out-side in H 2 O 2 signaling plays an important role in promoting vascular adaptation in T2D via Cys oxidation of redox-sensitive kinases required for endothelial function and angiogenesis in skeletal muscles.

In kidney transplantation acute allograft rejection is the most common cause of late allograft loss. Changes in indoleamine 2,3 dioxygenase (IDO) activity, which catabolizes the degradation of tryptophan to kynurenine, may predict rejection. However, exogenous IDO is immunosuppressive in rodent kidney transplantation. Thus, the increase in IDO activity observed in acute allograft rejection is insufficient to prevent rejection. To address this question, we assessed the regulation of IDO and its role in acute rejection in a porcine model of kidney transplant. In tissue samples from rejecting kidney allografts, we showed a 13-fold increase in IDO gene transcription and 20-fold increase in IDO enzyme activity when compared with autotransplanted kidneys. Allografts also demonstrated an over fourfold increase in tissue interferon (IFN)-γ, with marked increases in tumor necrosis factor (TNF)-α, TNF-β and interleukin 1β. Gene transcription and protein levels of kynurenine 3-monooxygenase (KMO) were decreased. KMO generates the immunosuppressive kynurenine, 3-hydroxykynurenine. The results of these studies demonstrate a clear association between rejection and increased allograft IDO expression, likely driven in part by IFN-γ and facilitated by other cytokines of the allogeneic response. Moreover, the loss of downstream enzymatic activity in the IDO metabolic pathway may suggest novel mechanisms for the perpetuation of rejection.

Pigs represent a desirable animal model for the study of rejection in kidney transplantation with inbred Yucatan miniature swine (YMS) the most commonly studied strain due to well defined swine leukocyte antigen (SLA) genotypes. However, limitations to YMS may include cost and availability. Outbred Yorkshire pigs are widely available and significantly cheaper than YMS. Recent advances in SLA genotyping have allowed its application to outbred strains. On this basis, we theorized that Yorkshire pigs would be a viable alternative to YMS for the study of rejection in kidney transplantation. To address this question, we performed auto (Auto) and allotransplants (Allo) in 24 Yorkshire pigs, and assessed SLA genotypes and acute rejection after 72h. At sacrifice, and when compared to autotransplants, allotransplants had significant elevations in serum creatinine (8.4±1.3 vs 2.8±2.0mg/dL for Allo vs autotransplants, respectively) and BUN (61±9 vs 19.2±15mg/dL for Allo vs autotransplants, respectively). Warm ischemia times between the two groups did not differ (24±2.3 vs 26.4±1.4min for Auto vs Allo, respectively). There were 16 distinct SLA haplotypes identified from pigs undergoing allotransplantion, no matched donor-recipient pairs, and all allografts demonstrated rejection. Type IIA cellular rejection (Banff) was the most common. One allograft demonstrated hyperacute rejection due a blood group incompatibility. Histologically, the expression of regulatory Tcells and dendritic cells was increased in allografts. These data suggest that Yorkshire pigs may be a useful model for the study of acute rejection in experimental kidney transplantation.