Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor alpha chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34(+)CD38(-) cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

Gamma delta (γδ) T cells are essential to protective immunity. In humans, most γδ T cells express Vγ9Vδ2+ T cell receptors (TCRs) that respond to phosphoantigens (pAgs) produced by cellular pathogens and overexpressed by cancers. However, the molecular targets recognized by these γδTCRs are unknown. Here, we identify butyrophilin 2A1 (BTN2A1) as a key ligand that binds to the Vγ9+ TCR γ chain. BTN2A1 associates with another butyrophilin, BTN3A1, and these act together to initiate responses to pAg. Furthermore, binding of a second ligand, possibly BTN3A1, to a separate TCR domain incorporating Vδ2 is also required. This distinctive mode of Ag-dependent T cell activation advances our understanding of diseases involving pAg recognition and creates opportunities for the development of γδ T cell–based immunotherapies.

The E2F family of transcription factors has been implicated in the regulation of cell proliferation, and £2F-binding sites are present in the promoters of several growth-regulating genes. E2F family members are functionally regulated, in part, by complex formation with one or more members of the nuclear pocket protein family, RB, pl07, and pl30. Pocket protein regulation of E2F likely contributes to normal cellular growth control. While the three cloned species of E2F, E2F-1, E2F-2, and E2F-3, are known to be targets of RB interaction, no E2F species has yet been shown to be a specific pl07 or pl30 target. Here, we describe the cloning of a new member of the E2F family, E2F-4, which forms heterodimers with a member(s) of the DP family and, unlike some family members, is present throughout the cell cycle and appears to be a differentially phosphorylated pl07-binding partner. pl07 binding not only can be linked to the regulation of E2F-4 transcriptional activity, but also to suppression of the ability of E2F-4 to transform an immortalized rodent cell line.

Little is known of the mechanisms controlling the Go/G1 transition of the cell cycle. The induction of immediate early gene expression, thought to be important for this process, suggests that the key factors controlling this transition preexist in quiescent cells. The E2F family of transcription factors likely play an important role in this process, because E2F DNA-binding activity exists in quiescent cells, and the induction of at least some immediate early genes requires intact E2F regulatory promoter sites. Here, we show that the major Go E2F activity of primary human T cells, E2F-4, is stably bound to the p130 pocket protein in association with a DP heterodimerization partner, p130-E2F-4 binding has functional implications because p130 effectively suppressed E2F-4-mediated trans-activation, and coexpression of E2F-4 overcame pl30-mediated G1 arrest more efficiently than RB-induced G1 blockade. Conversely, E2F-1 overrode an RB-induced G~ block more efficiently than E2F-4. Thus, p130 and RB appear to induce cell cycle arrest via biochemically distinct mechanisms that involve different E2F family members.

Independent of its antiapoptotic function, Bcl-2 can, through an undetermined mechanism, retard entry into the cell cycle. Cell cycle progression requires the phosphorylation by cyclin-dependent kinases (Cdks) of retinoblastoma protein (pRB) family members to free E2F transcription factors. We have explored whether retarded cycle entry is mediated by the Cdk inhibitor p27 or the pRB family. In quiescent fibroblasts, enforced Bcl-2 expression elevated levels of both p27 and the pRB relative p130. Bcl-2 still slowed G 1 progression in cells deficient in pRB but not in those lacking p27 or p130. Hence, pRB is not required, but both p27 and p130 are essential mediators. The ability of p130 to form repressive complexes with E2F4 is implicated, because the retardation by Bcl-2 was accentuated by coexpressed E2F4. A plausible relevant target of p130/E2F4 is the E2F1 gene, because Bcl-2 expression delayed E2F1 accumulation during G 1 progression and overexpression of E2F1 overrode the Bcl-2 inhibition. Hence, Bcl-2 appears to retard cell cycle entry by increasing p27 and p130 levels and maintaining repressive complexes of p130 with E2F4, perhaps to delay E2F1 expression.In addition to its well-established function in controlling cell survival, the Bcl-2 family (1) has been found to influence the cell cycle. Although Bcl-2 and its prosurvival relatives do not affect the growth rate in proliferating cultures, they can both accelerate withdrawal from the cycle (46) and retard reentry (4,29,30,35). Conversely, a shortened G 1 is found in lymphocytes that lack Bcl-2 (29) or express the Bcl-2 antagonist Bax (4). Thus, Bcl-2 appears to have a physiological role in influencing the transition between the quiescent and cycling states. That this ability is separate from its role in cell survival (46) is most clearly shown by mutations of Bcl-2 that eliminate its cell cycle activity but spare its antiapoptotic function (17,45).The cell cycle control function of Bcl-2 has ramifications for cellular homeostasis. Cycling cells are often more vulnerable to apoptosis, perhaps because, under conditions unfavorable for proliferation, certain cell cycle effectors promote apoptosis (11). Hence, promoting quiescence under conditions of stress may provide Bcl-2 with an additional, albeit indirect, means to enhance cell survival (30,46). Interference with Bcl-2's cell cycle effect may also augment its oncogenic role (see Discussion).Progression through the cell cycle requires the action of cyclin-dependent kinases (Cdks) (38, 39). As cells enter the cycle, newly synthesized D-type cyclins associate with and activate their Cdk-4/6 catalytic partners in mid-to late G 1 phase, while cyclin E appears later in G 1 and activates its Cdk-2 kinase subunit near the G 1 /S boundary. Opposing their activity are Cdk inhibitors (Cki) of two classes: INK4 proteins, such as p16, specifically inhibit D-cyclin kinases, whereas Cip/Kip proteins, such as p21 and p27, also inhibit Cdk-2 (38, 39). Other key negative regulators include the best-known Cdk substrates...

Coordinated interactions between cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors regulate orderly cell cycle progression. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylate the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending upon the cell type. In these studies overexpression of E2F-1 inhibited cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 were required for full repression of cyclin D1. Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter were required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at ؊127 bound Sp1, Sp3, and Sp4, and the distal region at ؊143 bound E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induced cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell.

Acute myeloid leukemia (AML) is a biologically heterogeneous group of related diseases in urgent need of better therapeutic options. Despite this heterogeneity, overexpression of the interleukin (IL)-3 receptor α-chain (IL-3 Rα/CD123) on both the blast and leukemic stem cell (LSC) populations is a common occurrence, a finding that has generated wide interest in devising new therapeutic approaches that target CD123 in AML patients. We report here the development of CSL362, a monoclonal antibody to CD123 that has been humanized, affinity-matured and Fc-engineered for increased affinity for human CD16 (FcγRIIIa). In vitro studies demonstrated that CSL362 potently induces antibody-dependent cell-mediated cytotoxicity of both AML blasts and CD34(+)CD38(-)CD123(+) LSC by NK cells. Importantly, CSL362 was highly effective in vivo reducing leukemic cell growth in AML xenograft mouse models and potently depleting plasmacytoid dendritic cells and basophils in cynomolgus monkeys. Significantly, we demonstrated CSL362-dependent autologous depletion of AML blasts ex vivo, indicating that CSL362 enables the efficient killing of AML cells by the patient's own NK cells. These studies offer a new therapeutic option for AML patients with adequate NK-cell function and warrant the clinical development of CSL362 for the treatment of AML.

Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI-and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of