Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia.

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence.

Cloning and characterisation of Ifi206: a new murine HIN-200 family member.

HIN-200 proteins are interferon-inducible proteins capable of regulating cell growth, senescence, differentiation and death. Using a combination of in silico analysis of NCBI EST databases and screening of murine C57BL/6 cDNA libraries we isolated novel murine HIN-200 cDNAs designated Ifi206S and Ifi206L encoding two putative mRNA splice variants. The p206S and p206L protein isoforms have a modular domain structure consisting of an N-terminal PAAD/DAPIN/Pyrin domain, a region rich in serine, threonine and proline residues and a C-terminal 200 B domain characteristic of other HIN-200 proteins. Ifi206 mRNA was detected only in the spleen and lung of BALB/c and C57BL/6 mice and expression was up-regulated by both types I and II IFN subtypes. p206 protein was predominantly expressed in the cytoplasm and addition of LMB, a CRM1 dependent nuclear export inhibitor, caused p206 to accumulate in the nucleus. Unlike other human and mouse HIN-200 proteins that contain only a single 200 amino acid domain, overexpression of p206 impaired the clonogenic growth of tumour cell lines. Thus, p206 represents the newest HIN-200 family member discovered. It has distinct and restricted pattern of expression however maintains many of the hallmarks of HIN-200 proteins including the presence of a characteristic 200 X domain, induction by interferon and an ability to suppress tumour cell growth.

G2/M checkpoint gene expression in developing germ cells.

Cell cycle progression is prevented by signal transduction pathways known as checkpoints which are activated in response to replication interference and DNA damage. We cloned a G2/M cell cycle phase-related checkpoint gene from a neonatal mouse testis cDNA library which was identified as mouse claspin, a proposed adaptor protein for Chk1. As part of a study on germ cell differentiation we examined the expression of the checkpoint gene, Chk1, and claspin at 12.5 and 14.5 days post coitum (dpc) and in the post-natal phase. Chk1 mRNA expression increased from 12.5 to 14.5 dpc in female gonads and was strong in males at both time points. Claspin however, was not detected until 14.5 dpc. This suggests there may be some dissociation of claspin expression from Chk1 in fetal germ cell development. Chk1 and claspin expression was also studied in testis over the first 3 days following birth, when apoptosis regulates germ stem cell number. We modulated checkpoint-related gene expression in testis using the anti-metabolite, 5-fluorouracil, resulting in increased apoptosis and upregulation of Chk1 (P<0.0001) and Cdc2 (P<0.02) mRNA. Although we do not fully understand the role checkpoint gene expression has during mammalian germ cell development this report is the first to show the expression of checkpoint-related genes in early mammalian germ cells.

Novel mechanisms of apoptosis induced by histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.

Suberoylanilide hydroxamic acid (SAHA) overcomes multidrug resistance and induces cell death in P-glycoprotein-expressing cells.

Multidrug resistance (MDR) mediated by the ATP-dependent efflux protein P-glycoprotein (P-gp) is a major obstacle to the successful treatment of many cancers. In addition to effluxing toxins, P-gp has been shown to protect tumor cells against caspase-dependent apoptosis mediated by Fas and tumor necrosis factor receptor (TNFR) ligation, serum starvation and ultraviolet (UV) irradiation. However, P-gp does not protect against caspase-independent cell death mediated by granzyme B or pore-forming proteins (perforin, pneumolysin and activated complement). We examined the effects of the chemotherapeutic hybrid polar compound suberoylanilide hydroxamic acid (SAHA) on P-gp-expressing MDR human tumor cell lines. In the CEM T-cell line, SAHA, a histone deacetylase inhibitor, induced equivalent death in P-gp-positive cells compared with P-gp-negative cells. Cell death was marked by the caspase-independent release of cytochrome c, reactive oxygen species (ROS) production and Bid cleavage that was not affected by P-gp expression. However, consistent with our previous findings, SAHA-induced caspase activation was inhibited in P-gp-expressing cells. These data provide evidence that P-gp inhibits caspase activation after chemotherapeutic drug treatment and demonstrates that SAHA may be of value for the treatment of P-gp-expressing MDR cancers.