T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here we describe a fratricide resistant ‘off-the-shelf’ CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide resistant, allo-tolerant ‘off-the-shelf’ CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin’s T cell lymphoma without a requirement for autologous T cells.

In human cancers, loss of PTEN, stabilization of HIF-1α and activation of Ras and AKT converge to increase the activity of a key regulator of glycolysis, 6-phosphofructo-2-kinase(PFKFB3). This enzyme synthesizes fructose 2,6-bisphosphate (F26BP) which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis. Previously, a weak competitive inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was found to reduce the glucose metabolism and proliferation of cancer cells. We have synthesized 73 derivatives of 3PO and screened each compound for activity against recombinant PFKFB3. One small molecule, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15), was selected for further pre-clinical evaluation of its pharmacokinetic, anti-metabolic and anti-neoplastic properties in vitro and in vivo. We found that PFK15 causes a rapid induction of apoptosis in transformed cells, has adequate pharmacokinetic properties, suppresses the glucose uptake and growth of Lewis lung carcinomas in syngeneic mice and yields anti-tumor effects in three human xenograft models of cancer in athymic mice that are comparable to FDA-approved chemotherapeutic agents. As a result of this study, asynthetic derivative and formulation of PFK15 has undergone IND-enabling toxicology and safety studies. A phase I clinical trial of its efficacy in advanced cancer patients will initiate in 2013 and we anticipate that this new class of anti-metabolic agents will yield acceptable therapeutic indices and prove to be synergistic with agents that disrupt neoplastic signaling.

IntroductionDeregulation of the proto-oncogene c-Myc (MYC) is considered one of a series of oncogenic events required for mammalian tumorigenesis. [1][2][3][4] MYC encodes a basic helix-loop-helix leucine zipper transcription factor that dimerizes with its partner, Max, and regulates multiple cellular functions including cell cycle, cell growth, differentiation, apoptosis, metabolism, and angiogenesis via transcription of downstream target genes. 2 The Myc/Max dimer can also repress transcription of another set of target genes through a less well-understood mechanism. 5,6 The c-Myc proto-oncogene is involved in transformation and cell proliferation in part via activation of the cyclin D2 promoter, 7 but also induces programmed cell death, mediated by nuclear respiratory factor 1 (NRF-1) 8 and the Arf-p53 pathway. 9,10 Forced expression of Myc in primary cells is generally thought to induce growth arrest or apoptosis. 11,12 The oncogenic function of c-Myc has been best studied in the E-Myc transgenic mouse, in which c-Myc expression is targeted to the lymphoid compartment by the immunoglobulin heavy chain gene promoter and enhancer. 13 In E-Myc mice, expression of Myc is not sufficient to cause leukemia. A latency period of 4 to 6 months is required for the accumulation of cooperating mutations before lymphoma can develop. 14 The majority of the E-Myc tumors harbor mutations in the now canonical Arf-Mdm2-p53 pathway. 14,15 Although the p53 pathway controls many functions including cell cycle, DNA damage response, and apoptosis, Bcl-2, or dominant-negative caspase 9 also cooperate with Myc, and alleviate the pressure to inactivate p53 during lymphomagenesis. 15 Therefore, of the many functions of the Arf-Mdm2-p53 pathway, it is the ability to mediate apoptosis that is targeted by cooperating mutations in Myc-induced lymphomagenesis.MYC dysregulation, via a variety of mechanisms, has also been associated with myeloid leukemias. 16 Double minute chromosomes in patients with acute myeloid leukemia (AML) contain MYC amplifications. 17,18 c-Myc expression is apparently required for the oncogenic effects of the Philadelphia chromosome product, BCL-ABL, 19 and overexpression of c-Myc also complements the transformation defects of BCR-ABL mutants. 20 A recent study showed that many important oncogenes in myeloid leukemogenesis including AML1-ETO, PML-RARA, and PLZF-RARA induce leukemogenesis by activating c-Myc, 21 suggesting c-Myc is a downstream target of these oncogenes. Lastly, c-Myc is upregulated by activating mutations in the gene encoding the FLT3 receptor tyrosine kinase, found in nearly one-third of all patients with AML. 22,23 There is a lack of useful animal models for studying the role of Myc in myeloid leukemia. AML infrequently develops in ESRMyc mice, in which a heterologous promoter inducibly drives Myc expression in bone marrow cells; however, most of these animals develop T-cell lymphomas, 24 making it a cumbersome system to Using the murine stem cell virus (MSCV) system to broadly express Myc in primar...