Haiyan Qin

Ponatinib suppresses the development of myeloid and lymphoid malignancies associated with FGFR1 abnormalities

Myeloid and lymphoid malignancies associated with fibroblast growth factor receptor-1 (FGFR1) abnormalities are characterized by constitutively activated FGFR1 kinase and rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. Molecular targeted therapies have not been widely used for stem cell leukemia/lymphoma (SCLL). Ponatinib (AP24534), which potently inhibits native and mutant BCR-ABL, also targets the FGFR family. Using murine BaF3 cells, stably transformed with six different FGFR1 fusion genes, as well as human KG1 cells expressing activated chimeric FGFR1 and five newly established murine SCLL cell lines, we show that ponatinib (<50 nM) can effectively inhibit phosphoactivation of the fusion kinases and their downstream effectors, such as PLCγ, Stat5 and Src. Ponatinib also significantly extended survival of mice transplanted with different SCLL cell lines. Ponatinib administered at 30 mg/kg daily also significantly delayed, or even prevented, tumorigenesis of KG1 cells in xenotransplanted mice. Furthermore, we demonstrate that ponatinib specifically inhibits cell growth and clonogenicity of normal human CD34+ progenitor cells transformed by chimeric FGFR1 fusion kinases. Overall, our data provide convincing evidence to suggest that pharmacologic inhibition of FGFR1 fusion kinases with ponatinib is likely to be beneficial for patients with SCLL and perhaps for other human disorders associated with dysregulated FGFR1 activity.

Src activation plays an important key role in lymphomagenesis induced by FGFR1-fusion kinases

Chromosomal translocations and activation of the fibroblast growth factor (FGF) receptor 1 (FGFR1) are a feature of stem cell leukemia-lymphoma syndrome (SCLL), an aggressive malignancy characterized by rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. It has been suggested that FGFR1 proteins lose their ability to recruit Src kinase, an important mediator of FGFR1 signaling, as a result of the translocations that delete the extended FGFR substrate-2 (FRS2) interacting domain that Src binds. In this study, we report evidence that refutes this hypothesis and reinforces the notion that Src is a critical mediator of signaling from the FGFR1 chimeric fusion genes generated by translocation in SCLL. Src was constitutively active in BaF3 cells expressing exogenous FGFR1 chimeric kinases cultured in vitro as well as in T-cell or B-cell lymphomas they induced in vivo. Residual components of the FRS2-binding site retained in chimeric kinases that were generated by translocation were sufficient to interact with FRS2 and activate Src. The Src kinase inhibitor dasatinib killed transformed BaF3 cells and other established murine leukemia cell lines expressing chimeric FGFR1 kinases, significantly extending the survival of mice with SCLL syndrome. Our results indicated that Src kinase is pathogenically activated in lymphomagenesis induced by FGFR1 fusion genes, implying that Src kinase inhibitors may offer a useful option to treatment of FGFR1-associated myeloproliferative/lymphoma disorders.

The WASF3-NCKAP1-CYFIP1 complex is essential for breast cancer metastasis

Inactivation of the WASF3 gene suppresses invasion and metastasis of breast cancer cells. WASF3 function is regulated through a protein complex that includes the NCKAP1 and CYFIP1 proteins. Here, we report that silencing NCKAP1 destabilizes the WASF3 complex, resulting in a suppression of the invasive capacity of breast, prostate, and colon cancer cells. In an in vivo model of spontaneous metastasis in immunocompromized mice, loss of NCKAP1 also suppresses metastasis. Activation of the WASF protein complex occurs through interaction with RAC1, and inactivation of NCKAP1 prevents the association of RAC1 with the WASF3 complex. Thus, WASF3 depends on NCKAP1 to promote invasion and metastasis. Here, we show that stapled peptides targeting the interface between NCKAP1 and CYFIP1 destabilize the WASF3 complex and suppress RAC1 binding, thereby suppressing invasion. Using a complex-disrupting compound identified in this study termed WANT3, our results offer a mechanistic proof of concept to target this interaction as a novel approach to inhibit breast cancer metastasis. Cancer Res; 76(17); 5133-42. ©2016 AACR.

Transformation of human CD34+ hematopoietic progenitor cells with DEK-NUP214 induces AML in an immunocompromised mouse model

Acute myeloid leukemia (AML) is a heterogeneous disease comprising a large number of subtypes defined by specific chromosome abnormalities. One such subtype carries the t(6;9)(p22;q34) chromosome rearrangement, which leads to expression of the DEK-NUP214 chimeric gene, and has a particularly poor outcome. To provide a better understanding of the molecular etiology of these relatively rare individual AML variants, it is necessary to generate in vivo models, which can also serve as a means to evaluate targeted therapies based on their specific genetic abnormalities. Here, we describe the development of a human cell AML, generated in CD34+ human hematopoietic progenitor cells xenografted into immunocompromised mice that express human myeloid cell growth factors. Within 6 months, these mice develop a human cell AML with phenotypic characteristics of the primary t(6;9) disease and a CD45+CD13+CD34+CD38+ immunophenotype. Gene expression studies show that members of the HOX family of genes (HOXA9, 10, B3, B4 and PBX3) are highly upregulated in the AML from this mouse model as well as from primary human t(6;9) AML. Gene expression analysis also identified several other significantly disregulated pathways involving KRAS, BRCA1 and ALK, for example. This is the first report of a humanized model of the DEK-NUP214 disease and provides a means to study the development and treatment of this particular subtype of AML.

Dysregulated signaling pathways in the development of CNTRL-FGFR1–induced myeloid and lymphoid malignancies associated with FGFR1 in human and mouse models

Myeloid and lymphoid neoplasm associated with FGFR1 is an aggressive disease, and resistant to all the current chemotherapies. To define the molecular etiology of this disease, we have developed murine models of this disease, in syngeneic hosts as well as in nonobese diabetic/severe combined immunodeficiency/interleukin 2Rγ(null) mice engrafted with transformed human CD34+ hematopoietic stem/progenitor cells. Both murine models mimic the human disease with splenohepatomegaly, hypercellular bone marrow, and myeloproliferative neoplasms that progresses to acute myeloid leukemia. Molecular genetic analyses of these model mice, as well as primary human disease, demonstrated that CNTRL-FGFR1, through abnormal activation of several signaling pathways related to development and differentiation of both myeloid and T-lymphoid cells, contribute to overt leukemogenesis. Clonal evolution analysis indicates that myeloid related neoplasms arise from common myeloid precursor cells that retain potential for T-lymphoid differentiation. These data indicate that simultaneously targeting these pathways is essential to successfully treating this almost invariably lethal disease.

Development of ZMYM2‐FGFR1 driven AML in human CD34+ cells in immunocompromised mice

Acute myelogenous leukemia (AML) has an overall poor survival rate and shows considerable molecular heterogeneity in its etiology. In the WHO classification there are >50 cytogenetic subgroups of AML, many showing highly specific chromosome translocations that lead to constitutive activation of individual kinases. In a rare stem cell leukemia/lymphoma syndrome, translocations involving 8p11 lead to constitutive activation of the fibroblast growth factor receptor 1 (FGFR1) kinase. This disorder shows myeloproliferative disease with almost invariable progresses to AML and conventional therapeutic strategies are largely unsuccessful. Because of the rare nature of this syndrome, models that faithfully recapitulate the human disease are needed to evaluate therapeutic strategies. The t(8;13)(p11;q12) chromosome translocation is most common rearrangement seen in this syndrome and creates a ZMYM2‐FGFR1 chimeric kinase. To understand more about the molecular etiology of AML induced by this particular rearrangement, we have created a model human CD34+ cells transplanted into immunocompromized mice which develop myeloproliferative disease that progresses to AML with a long (>12 months) latency period. As in humans, these mice show hepatospenomegaly, hypercellular bone marrow and a CD45 + CD34 + CD13+ immunophenotype. Molecular studies demonstrate upregulation of genes such as KLF4 and FLT3 that promote stemness, and overexpression of MYC, which is associated with suppression of myeloid cell differentiation. This murine model, therefore, provides an opportunity to develop therapeutic strategies against the most common subtype within these FGFR1 driven neoplasms and study the molecular etiology in more depth.

A model of BCR-FGFR1 driven human AML in immunocompromised mice

Carlile, G.W., Smith, D.H. & Wiedmann, M. (2004) Caspase-3 has a nonapoptotic function in erythroid maturation. Blood, 103, 4310–4316. Centis, F., Tabellini, L., Lucarelli, G., Buffi, O., Tonucci, P., Persini, B., Annibali, M., Emiliani, R., Iliescu, A., Rapa, S., Rossi, R., Ma, L., Angelucci, E. & Schrier, S.L. (2000) The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major. Blood, 96, 3624– 3629. Dai, M.-S., Mantel, C.R., Xia, Z.-B., Broxmeyer, H.E. & Lu, L. (2000) An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2. Blood, 96, 3985–3987. Fibach, E. & Rachmilewitz, E. (1993) Stimulation of erythroid progenitors by high concentrations of erythropoietin results in normoblasts arrested in G2 phase of the cell cycle. Experimental Hematology, 21, 184–188. Forster, L., McCooke, J., Bellgard, M., Joske, D., Finlayson, J. & Ghassemifar, R. (2015) Differential gene expression analysis in early and late erythroid progenitor cells in b-thalassaemia. British Journal of Haematology, 170, 257–267. Hristoskova, S., Holzgreve, W. & Hahn, S. (2003) Fetal nucleated erythrocytes in maternal circulation do not display a classic membrane-associated apoptotic characteristic (phosphatidylserine exposure) despite being positive by terminal dUTP nuclear end labeling. Clinical Chemistry, 49, 1934–1937. Libani, I.V., Guy, E.C., Melchiori, L., Schiro, R., Ramos, P., Breda, L., Scholzen, T., Chadburn, A., Liu, Y., Kernbach, M., Baron-Luhr, B., Porotto, M., de Sousa, M., Rachmilewitz, E.A., Hood, J.D., Cappellini, M.D., Giardina, P.J., Grady, R.W., Gerdes, J. & Rivella, S. (2008) Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia. Blood, 112, 875–885. Mathias, L.A., Fisher, T.C., Zeng, L., Meiselman, H.J., Weinberg, K.I., Hiti, A.L. & Malik, P. (2000) Ineffective erythropoiesis in beta-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage. Experimental Hematology, 28, 1343–1353. Wojda, U., Noel, P. & Miller, J.L. (2002) Fetal and adult hemoglobin production during adult erythropoiesis: coordinate expression correlates with cell proliferation. Blood, 99, 3005–3013. Yuan, J., Angelucci, E., Lucarelli, G., Aljurf, M., Snyder, L.M., Kiefer, C.R., Ma, L. & Schrier, S.L. (1993) Accelerated programmed cell death (apoptosis) in erythroid precursors of patients with severe beta-thalassemia (Cooley’s anemia). Blood, 82, 374–377.

Mutation in the FGFR1 tyrosine kinase domain or inactivation of PTEN is associated with acquired resistance to FGFR inhibitors in FGFR1-driven leukemia/lymphomas

Stem cell leukemia/lymphoma syndrome (SCLL) is driven by constitutive activation of chimeric FGFR1 kinases generated by chromosome translocations. We have shown that FGFR inhibitors significantly suppress leukemia and lymphoma development in vivo, and cell viability in vitro. Since resistance to targeted therapies is a major reason for relapse, we developed FGFR1‐overexpressing mouse and human cell lines that are resistant to the specific FGFR inhibitors AZD4547 and BGJ398, as well as non‐specific inhibitors, such as ponatinib, TKI258 and E3810. Two mutually exclusive mechanisms for resistance were demonstrated; an activating V561M mutation in the FGFR1 kinase domain and mutational inactivation of PTEN resulting in increased PI3K/AKT activity. Ectopic expression of PTEN in the PTEN‐mutant cells resensitizes them to FGFR inhibitors. Treatment of resistant cells with BGJ398, in combination with the BEZ235 PI3K inhibitor, shows an additive effect on growth in vitro and prolongs survival in xenograft models in vivo. These studies provide the first direct evidence for both the involvement of the FGFR1 V561M mutation and PTEN inactivation in the development of resistance in leukemias overexpressing chimeric FGFR1. These studies also provide a potential strategy to treat leukemias and lymphomas driven by FGFR1 activation that become resistant to FGFR1 inhibitors.

Targeting FGFR1 to suppress leukemogenesis in syndromic and de novo AML in murine models

Although over expression of chimeric FGFR1 kinase consistently leads to the development of AML in the rare Stem Cell Leukemia and Lymphoma syndrome, we now show that overexpression of FGFR1 is also seen in up to 20% of non-syndromic, de novo AML. To determine whether targeting FGFR1 in both of these AML subtypes can suppress leukemogenesis, we evaluated the effects of different FGFR1 inhibitors in a side-by-side comparison for their ability to affect in vitro proliferation in FGFR1 overexpressing murine and human cells lines. Three newly developed pan-FGFR inhibitors, AZD4547, BGJ398 and JNJ42756493, show a significantly improved efficacy over the more established FGFR inhibitors, PD173074 and TKI258. To examine whether targeting FGFR1 suppresses leukemogenesis in de novo AML in vivo, we created xenografts in immunocompromized mice from primary, de novo AML that showed > 3-fold increased expression of FGFR1. Using BGJ398, the most potent inhibitor identified in the in vitro studies, AML progression in these mice was significantly suppressed compared with vehicle treated animals and overall survival improved. Importantly, no difference in disease course or survival was seen in AML xenografts that did not show overexpression of FGFR1. These observations support the idea that FGFR1 is a driver oncogene in de novo, FGFR1-overexpressing AML and that molecularly targeted therapies using FGFR1 inhibitors may provide a valuable therapeutic regimen for all FGFR1-overexpressing AML.

FGFR1OP2-FGFR1 induced myeloid leukemia and T-cell lymphoma in a mouse model

Myeloproliferative neoplasms (MPN) associated with chromosome translocations involving the fibroblast growth factor receptor 1 (FGFR1) gene is a distinct disease entity in the current WHO classification1,2 (hereafter MPN-tFGFR1). These patients have a poor outcome with a 5-year survival rate of < 20%. MPN-tFGFR1 patients typically present with bilineage disease (myeloid and lymphoid) and frequently (~80%) progress to AML.3 There have been at least 15 different chromosome rearrangements described, which have been shown to or are predicted to lead to ligand-independent, constitutive activation of the FGFR1 kinase.3 Even though activation of FGFR1 is seen in all cases, there is variability in the clinical presentation of the disease, depending on the specific chromosome translocations that are present.3 Because this is a rare disease, developing better therapies requires models that show the same phenotypic and genetic characteristics. One aggressive variant of this disease shows a t(8;12)(p12;p11) chromosome translocation that generates the chimeric FGFR1OP2-FGFR1 protein.4 We have used a mouse bone marrow transduction and transplantation approach to develop a model for FGFR1OP2-FGFR1 disease where, consistent with the human disease, the mice concurrently developed rapid onset CD4+ T-cell lymphoblastic lymphoma and acute myeloid leukemia (cKit+Gr1+Mac1+CD19+). Molecular analysis demonstrates that the T-cell lymphomas are associated with activating mutations of Notch1, and the progression of AML is associated with dysregulation of genes involved in multiple signaling pathways related to myeloid cell development and differentiation, including KIT, CSF1R, SPI1, IRF8 and KLF4. This highly representative mouse model will allow evaluation of potential therapies against this almost invariably lethal disease.