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Figure S1. Skin exposure to UVB induces splenic erythroid cell proliferation in mice. Figure S2. Skin exposure to UVB results in expansion of splenic cell populations with erythroid colony-forming potential. Figure S3. Administration of an anti-EPO antibody prevents UVB-responsive splenic erythroid expansion. Figure S4. Tumor growth induces splenic erythroid cell proliferation in mice. Figure S5. CD235A immunostaining identifies tumor-associated erythroid cells in human cancer tissues.

Hematopoietic-derived cells are integral components of the tumor microenvironment and serve as critical mediators of tumor–host interactions. Host cells derived from myeloid and lymphoid lineages perform well-established functions linked to cancer development, progression, and response to therapy. It is unclear whether host erythroid cells also contribute to shaping the path that cancer can take, but emerging evidence points to this possibility. Here, we show that tumor-promoting environmental stress and tumor-induced hemodynamic changes trigger renal erythropoietin production and erythropoietin-dependent expansion of splenic erythroid cell populations in mice. These erythroid cells display molecular features indicative of an immature erythroid phenotype, such as the expression of both CD71 and TER119 and the retention of intact nuclei, and express genes encoding immune checkpoint molecules. Nucleated erythroid cells with similar properties are present in mouse and human tumor tissues. Antibody-mediated erythropoietin blockade reduces tumor-responsive erythroid cell induction and tumor growth. These findings reveal the potential of tumor-induced erythropoietin and erythroid cells as targets for cancer treatment.Implications: Our study identifies erythropoietin and erythroid cells as novel players in tumor–host interactions and highlights the involvement of multiorgan signaling events in their induction in response to environmental stress and tumor growth.

Deletions and dominant-negative mutations in IKZF1, the gene encoding Ikaros transcription factor, are found in ∼85% of Ph+ B-ALL and in some cases of Ph− B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph− or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and ponatinib have been incorporated into chemotherapy regimens for Ph+ B-ALL, the majority of patients still relapse, which correlates with residual bone marrow disease following induction therapy. New therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph− B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) that mimics the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated that loss of Ikaros DNA-binding function arrests B-lymphoid development at a large pre-B cell stage that can give rise to B-ALL. Survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK downstream of integrins and growth factor receptors that plays important roles in cancer stem cell biology and the tumor microenvironment. Here, we show that dysregulated tyrosine kinases, including BCR-ABL1, cooperate with Ikaros mutation to accelerate the development of B-lymphoblastic leukemia in mice, correlated with a striking (∼30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Ikzf1-mutant BCR-ABL1+ lymphoblasts exhibit relative resistance to ABL1 TKIs including dasatinib that is dependent on the presence of stroma. VS-4718 is a potent, orally bioavailable FAK inhibitor that inhibits tumor growth and metastasis in preclinical models, and is currently under evaluation in clinical trials in patients with various solid tumors. VS-4718 treatment abolished stromal adhesion and induced apoptosis of Ikzf1-mutant B-ALL and synergized with dasatinib, but had little effect on Ikzf1 WT B-ALL cells. Primary human Ph+ B-ALL samples showed a correlation between IKZF1 mutation status, stromal adherence, and sensitivity to FAK and ABL inhibitors in vitro, and BM tropism in vivo in xenografted NSG mice. Results from ongoing in vivo therapy experiments will be presented. Together, these results suggest a new model for the pathogenesis of high-risk B-ALL and its resistance to conventional therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM leukemic stem cells. Inhibition of FAK signaling in Ph+ or Ph− IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. FAK inhibitors represent a promising new approach for the treatment of high-risk IKZF1-mutant B-ALL patients. Citation Format: Nilamani Jena, Ila Joshi, Toshimi Yoshida, Zhihong Zhang, Zhong-Ying Liu, Prasanthi Tata, Irina M. Shapiro, Jonathan A. Pachter, David T. Weaver, Katia Georgopoulos, Richard A. Van Etten. Targeting focal adhesion kinase is a novel approach to therapy of high-risk, Ikaros-mutant acute B-cell lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2015-4202