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Background 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. Methods Stem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq was used to examine the related signaling pathways and regulated target genes. Results For the first time, we show that this non-classical truncated form of FGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells in an animal model in vivo. 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. 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. We further show that de novo human AML also express 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. Combined treatment with FGFR1 and FLT3 inhibitors provides increased survival compared to FGFR1 inhibition alone. Conclusions This study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by the truncated nuclear tnFGFR1 derivative, 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 an 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.

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.

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.