Weissman, Irving L.

Abstract Introduction Polycythemia vera (PV) is characterized by excessive production of erythroid cells and in most cases a point mutation (V617F) in the Jak2 cytokine signaling kinase. We investigated whether a selective JAK2 inhibitor decreased Jak2 V617F induced erythroid differentiation. Methods Wild-type and mutant Jak2 V617F genes were excised from the retroviral Jak2-mus-MSCV-neo vector (Levine et al), cloned into the lentiviral vector pLV CMV IRES2 GFP and their presence verified by DNA sequencing. Lentiviral vectors bearing the wild-type and mutant Jak2 genes or vector alone were used to transduce human peripheral blood CD34+ cells, which were then divided for plating into megacult medium for megakaryocytic colony growth and methylcellulose culture for enumeration of all other progenitor cell types. Normal cord blood HSC (CD34+/CD38−/CD90+) were clone sorted with the FACS Aria and transduced with no vector, backbone vector, wild-type JAK2 or mutant JAK2 vector in methocult media (Stem Cell Technologies Inc, GF+ H4435) +/− 300 nM of a selective JAK2 inhibitor, TG101348. Colonies were scored at day 14. RNA was isolated from the colonies (Qiagen RNeasy kit) and RT-PCR was performed with wild-type and mutant JAK2 allele specific primers. Results Transduction of cord blood HSC with the mutant Jak2 vector resulted in skewed erythroid colony formation compared to wild-type Jak2, vector alone and untransduced HSC (Figure 1; n=3). RT-PCR with murine Jak2 specific primers resulted in ~900 bp fragments corresponding to murine Jak2 from colonies transduced with the wild-type and mutant Jak2 and confirmed by sequencing, but not those from colonies transduced with the vector alone or the untransduced cells. Like the results in cord blood cells, adult peripheral blood CD34+ cells transduced with the mutant Jak2 developed a skewed developmental pattern, with far greater erythroid colony formation compared to wild-type Jak2 or vector alone. In megacult assays, CD34+ cells transduced with the mutant Jak2 had similar megakaryocytic potential as wild-type Jak2 or vector alone. Addition of TG101348 (300 nM), inhibited mutant kinase-induced erythroid colony formation (Figure 1) in 3 experiments while 100– 300 nM was inhibitory to PV (n=2 patients) HSC and progenitors. Current experiments focus on inhibition of Jak2 in a bioluminescent highly immunocompromised mouse model of Jak2V617F-induced myeloproliferation (Figure 2). Conclusion JAK2 V617F skews differentiation of HSC toward the erythroid lineage and may be inhibited with a selective JAK2 inhibitor - TG101348. Figure 1. In vitro JAK2 Inhibition. Figure 1. In vitro JAK2 Inhibition. Figure 2. Bioluminescent JAK2 V617F-induced Myeloproliferation Model. Figure 2. Bioluminescent JAK2 V617F-induced Myeloproliferation Model.

Abstract Glioblastoma is the most common primary tumor of the CNS in adults, representing approximately 50% of all gliomas and 15% of primary brain tumors. The current standard of care for GBM is safe maximal surgical resection followed by radiotherapy and concurrent Temozolomide (TMZ), but median survival continues to be 15–16 months. Temozolomide is a second-generation DNA alkylating agent that induces thymine mispairing during DNA replication resulting in tumor cell G2/M phase arrest and autophagy, a standard care of therapy against GBM. While the success of TMZ in clinical trials showed great promise for its overall efficacy, emerging TMZ resistance make us to think more for the combination therapy. CD47, a tumor cell surface marker, plays as “don't eat me signal” through binding its receptor SIRPα on macrophages and the antibody against CD47, which blocks interactions of CD47 with SIRPα, has been shown to lead to tumor destruction. Furthermore, CD47 is a prognostic marker as its expression predisposes cancer patients to a poorer survival outcome. This has significant clinical implications since approximately more than 80% of patients with the most GBMs, overexpress CD47. Overexpression of CD47 is also associated with a decreased probability of survival in clinical cohorts of GBM. Our work and that of others demonstrate that CD47 blockade enables tumor cell phagocytosis by antigen presenting cells (APC), establishing this molecule as a viable therapeutic target in GBM. We additionally investigated the combinatorial effect of CD47 blockade with temozolomide. DNA alkylating agents induce sporadic tumor cell necrosis associated with the extracellular release of damage associated molecular patterns (DAMPs) such as calreticulin, a pro-phagocytic protein whose interaction with low density lipoprotein receptor-related protein 1 (LRP1) facilitates recognition by professional antigen-presenting cells (APCs) and acts as a critical molecular component in promoting immunogenic cell. Our results showed that tumor cell treatment with temozolomide induces plasma membrane expression of calreticulin. Combination therapy resulted in amplified tumor cell phagocytosis and antigen presentation by APC. In addition, preclinical assessment of combination therapy in a syngeneic murine model of GBM resulted in significantly improved survival, characterized by increased intra-tumor penetration of APC cells. These results suggest that the combination of CD47 blockade with temozolomide may enhance tumor immunogenicity, and can improve clinical outcomes demonstrated by mono-therapeutic approaches with conventional chemotherapy. Citation Format: Yaqing Qie, Christina Von Roemeling, Yuanxin Chen, Kevin Shih, Xiujie Liu, Wen Jiang, Joshua Knight, Charles Chan, Irving Weissman, Betty Kim. CD47 blockade with temozolomide can enhance the therapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-208. doi:10.1158/1538-7445.AM2017-LB-208