Acquired aplastic anemia is an autoimmune-mediated bone marrow failure syndrome. The mechanism by which such an autoimmune reaction is initiated is unknown. Whether and how the genetic lesions detected in patients cause autoimmune bone marrow failure have not yet been determined. We found that mice with spontaneous deletion of the TGFβ-activated kinase-1 gene in a small subset of hematopoietic cells developed bone marrow failure which resembled the clinical manifestations of acquired aplastic anemia patients. Bone marrow failure in such mice could be reversed by depletion of CD4+ T lymphocytes or blocked by knockout of interferon-γ, suggesting a Th1-cell-mediated autoimmune mechanism. The onset and progression of bone marrow failure in such mice were significantly accelerated by the inactivation of tumor necrosis factor-α signaling. Tumor necrosis factor-α restricts autoimmune bone marrow failure by inhibiting type-1 T-cell responses and maintaining the function of myeloid-derived suppressor cells. Furthermore, we determined that necroptosis among a small subset of mutant hematopoietic cells is the cause of autoimmune bone marrow failure because such bone marrow failure can be prevented by deletion of receptor interacting protein kinase-3. Our study suggests a novel mechanism to explain the pathogenesis of autoimmune bone marrow failure.

Acquired aplastic anemia (AAA) is an autoimmune-mediated bone marrow failure (BMF) syndrome. Cryptic clonal genetic lesions were commonly detected in a small subset of hematopoietic stem/progenitor cells (HSPCs) in most patients' BM samples. The mechanism by which the autoimmune reaction is initiated is unknown. Whether and how these cryptic clonal genetic lesions might cause autoimmune BMF have not yet been determined. We found that mice with spontaneous deletion of the TGFβ-activated kinase-1 (Tak1) gene in a small subset of HSPCs (1-3%) developed BMF which resembled the clinical manifestations of AAA patients. BMF in such mice could be reversed by depletion of CD4+ T lymphocytes or treatment with IFN-γ, suggesting a Th1 cell-mediated autoimmune mechanism. Interestingly, the disease onset and progression of BMF in such mice were significantly accelerated by inactivation of TNF-α signaling, indicating that TNF-α might restrict the progression of autoimmune BMF. Furthermore, we determined that the necroptosis of a small subset of hematopoietic cells is the cause of autoimmune BMF because such BMF can be completely prevented by deletion of Rip3, a key necroptotic mediator. Our study suggested that the necroptosis of a small subset of hematopoietic cells induces autoimmune BMF, and that elevated TNF-α restricts the progression of such autoimmune BMF. We believe that, in addition to inhibiting T-cell-mediated autoimmune reactions to induce disease remission, repression of the necroptosis of mutant HSPCs might be necessary to prevent disease relapse and progression of autoimmune BM failure. Disclosures Stiff: Gilead: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Fate Therapeutics: Research Funding; Plasmacyclics: Consultancy, Honoraria, Research Funding; Eisai: Research Funding.

Mutations and inactivation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) are observed in 15%–25% of cases of human T cell acute lymphoblastic leukemia (T-ALL). Pten deletion induces myeloproliferative disorders (MPDs), acute myeloid leukemia (AML), and/or T-ALL in mice. Previous studies attributed Pten-loss-related hematopoietic defects and leukemogenesis to excessive activation of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. Although inhibition of this signal dramatically suppresses the growth of PTEN-null T-ALL cells in vitro, treatment with inhibitors of this pathway does not cause a complete remission in vivo. Here, we report that focal adhesion kinase (Fak), a protein substrate of Pten, also contributes to T-ALL development in Pten-null mice. Inactivation of the FAK signaling pathway by either genetic or pharmacologic methods significantly sensitizes both murine and human PTEN-null T-ALL cells to PI3K/AKT/mTOR inhibition when cultured in vitro on feeder layer cells or a matrix and in vivo.

Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK–AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF–JNK–AP1 signaling pathway. Our data suggest that co-inhibition of both TNF–JNK–AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

Tumor necrosis factor-α (TNF-α)-induced RIP1/RIP3 (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3)-mediated necroptosis has been proposed as an alternative strategy for treating apoptosis-resistant leukemia. However, we found that most acute myeloid leukemia (AML) cells, especially M4 and M5 subtypes, produce TNF and show basal level activation of RIP1/RIP3/MLKL signaling, yet do not undergo necroptosis. TNF, through RIP1/RIP3 signaling, prevents degradation of SOCS1, a key negative regulator of interferon-γ (IFN-γ) signaling. Using both pharmacologic and genetic assays, we show here that inactivation of RIP1/RIP3 resulted in reduction of SOCS1 protein levels and partial differentiation of AML cells. AML cells with inactivated RIP1/RIP3 signaling show increased sensitivity to IFN-γ-induced differentiation. RIP1/RIP3 inactivation combined with IFN-γ treatment significantly attenuated the clonogenic capacity of both primary AML cells and AML cell lines. This combination treatment also compromised the leukemogenic ability of murine AML cells in vivo. Our studies suggest that inhibition of RIP1/RIP3-mediated necroptotic signaling might be a novel strategy for the treatment of AML when combined with other differentiation inducers.

Leukemic Stem Cells (LSCs) isolated from Acute Myeloid Leukemia (AML) patients are highly sensitive to NF-κB inhibition-induced cell death in in vitro culture when compared to normal hematopoietic stem/progenitor cells (HSPCs). This suggests blocking NF-κB could be an effective strategy for treating AML. However, NF-κB inhibitor treatment alone is unable to clear AML tumors in vivo. We propose this is most likely due to elements within the niche microenvironment stimulating compensatory survival/proliferation signals in AML cells that can overcome NF-κB inhibition.

Abstract Abstract 1878 NF-κB activation is essential for leukemic cell and stem cell (LSC) survival and self-renewal, but is significantly less essential for similar functions in normal bone marrow hematopoietic stem/progenitor cells (HSPCs). As a result, LSCs are more sensitive to both pharmacologic and genetic NF-κB inhibition than HSPCs. These sensitivities suggest that NF-κB signaling could be a potential therapeutic target in the treatment of leukemia. However, high doses of NF-κB inhibitor treatment are also associated with significant inflammation-mediated toxicity to liver, skin and other tissues. Therefore, new approaches are needed that will be able to protect normal tissues while simultaneously enhancing the effects of NF-κB inhibition on leukemic cells. By utilizing genetic knock-out HSPC/leukemia models in combination with small molecule inhibitors, we searched for factors that could sensitize leukemic cells to NF-κB inhibition while simultaneously protecting HSPCs. We demonstrated that targeted inhibition of TNFα induced NF-κB-independent signaling would be a useful approach to treat leukemia in combination with NF-κB inhibition. We found that deactivating TNFα signaling either by genetic deletion of its receptors or through neutralizing the ligand with an antibody can significantly enhance NF-κB inhibition-induced leukemic cell elimination. In contrast, deactivation of TNFα signaling can significantly protect normal HSPCs from NF-κB inhibitor-induced death. Mechanistic studies revealed that TNFα stimulates several similar signals in both leukemic cells and HSPCs, including NF-κB, ERK, AKT, p38 and JNK. In order to determine which of these signals would best augment NF-κB inhibition, we performed biochemical analyses and searched for candidate survival signals activated downstream of TNFα and that operated independently of NF-κB. This analysis revealed that TNFα-induced ERK and AKT signals are NF-κB dependent, while TNFα-induced p38 and JNK signals are NF-κB independent. Inhibition of p38 enhanced leukemic cell growth, and was therefore ruled out as a candidate. Our analyses showed that JNK was activated by TNFα stimulation, operated independently of NF-κB activation, and also repressed leukemic cell growth. Further study confirmed that TNFα-dependent JNK activation has opposite functions in HSPCs and leukemic cells: JNK acts by promoting cell survival in leukemic cells while inducing cell death in HSPCs. We confirmed this result by inactivating the JNK signal via small molecule inhibitor, and found that we could significantly sensitize leukemic cells to NF-κB inhibition while protecting normal HSPCs from TNFα-mediated cell death associated with NF-κB inhibition. Mechanism analysis suggested that TNFα represses the growth of HSPCs by a JNK-RIP1/RIP3-dependent necroptosis mechanism, whereas TNFα promotes the expansion of leukemic cells by inducing the parallel activation of NF-κB-dependent AKT/ERK signaling and NF-κB-independent JNK signaling. In conclusion, our studies suggest the simultaneous inhibition of both NF-κB and TNFα-induced NF-κB-independent signals like JNK might provide a more comprehensive approach for targeted treatment of leukemias that also protects against deleterious inflammation in the bone marrow and other tissues. Disclosures: Nand: Celgene: Research Funding.

Abstract 864 Phosphatase and tensin homolog on chromosome 10 (Pten) is a tumor suppressor which possesses both lipid and protein phosphatase activities. Mutations and epigenetic inactivations of the Pten gene are commonly detected in a large number of tissue malignancies, including leukemias and lymphomas. Studies using Hematopoietic Pten -knockout in adult mice ( Pten −/− ) have demonstrated that Pten plays a critical role in maintaining the homeostasis of bone marrow (BM) hematopoiesis. Pten inactivation promotes the proliferation and peripheral mobilization of BM hematopoietic stem cells (HSCs). Pten −/− mice develop myeloproliferative disorders (MPD) within days, followed by acute leukemic transformation. Most previous studies attributed such phenotypic changes observed in Pten −/− mice to excessive activation of the PI3K/AKT/mTOR signal, a consequence of the loss of Pten9s lipid phosphatase activity. However, the role of Pten9s protein phosphatase activity in the regulation of HSCs and leukemogenesis is not well studied. Focal adhesion kinase (Fak) is a critical substrate for the protein phosphatase activity of Pten. Dysregulation of Fak has been observed in many cancers, including acute myeloid leukemias (AML) and acute lymphocytic leukemias (ALL). Therefore, we postulated that Fak might play a pivotal role in the development and progression of leukemia following Pten deletion. To test this hypothesis, we generated Mx1-Cre + Pten fl/fl Fak fl/fl mice (an interferon-inducible Pten and Fak compound-knockout, Pten −/− Fak −/− ) in which both the Pten and Fak genes in the hematopoietic system are deleted upon injection of polyinosinic-polycytidylic acid (pI-pC). Our results showed that the genetic inactivation of Fak can partially rescue HSC defects associated with Pten deficiency. We found that peripheral mobilization of HSCs in Pten −/− Fak −/− mice is significantly reduced compared to Pten −/− mice. As a consequence, more long-term HSCs (LT-HSCs) are preserved in the BM of Pten −/− Fak −/− mice compared to Pten −/− mice. Transplantation studies suggested that the hematopoietic reconstitutive capacity of Pten −/− Fak −/− HSCs is significantly improved compared to Pten −/− HSCs. Although Fak deletion fails to prevent the development of MPD in Pten −/− mice, Fak deletion does significantly reduce the frequency of AML/ALL, also significantly delays the onset of AML/ALL in comparison to Pten −/− mice. This study suggests that Fak might be a potential target for preventing the MPD-to-AML/ALL transformation and therefore blocking the Fak activity may hold a promise for a novel anti-leukemia therapy. The molecular mechanisms underlying the phenotype restoration of Pten −/− mice by Fak deletion in the hematopoietic system are actively being studied in our laboratory. Disclosures: No relevant conflicts of interest to declare.