Persistence of T cells engineered with chimeric antigen receptors (CARs) has been a major barrier to use of these cells for molecularly targeted adoptive immunotherapy. To address this issue, we created a series of CARs that contain the TCR-ζ signal transduction domain with the CD28 and/or CD137 (4-1BB) intracellular domains in tandem. After short-term expansion, primary human T cells were subjected to lentiviral gene transfer, resulting in large numbers of cells with >85% CAR expression. In an immunodeficient mouse xenograft model of primary human pre-B-cell acute lymphoblastic leukemia, human T cells expressing anti-CD19 CARs containing CD137 exhibited the greatest anti-leukemic efficacy and prolonged (>6 months) survival in vivo, and were significantly more effective than cells expressing CARs containing TCR-ζ alone or CD28-ζ signaling receptors. We uncovered a previously unrecognized, antigen-independent effect of CARs expressing the CD137 cytoplasmic domain that likely contributes to the enhanced antileukemic efficacy and survival in tumor bearing mice. Furthermore, our studies revealed significant discrepancies between in vitro and in vivo surrogate measures of CAR efficacy. Together these results suggest that incorporation of the CD137 signaling domain in CARs should improve the persistence of CARs in the tumor microenvironment and hence maximize their antitumor activity.

Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSCs). To validate this hypothesis in vivo, we developed a clinically relevant chemotherapeutic approach treating patient-derived xenograft (PDX) with cytarabine. Cytarabine residual AML cells are enriched neither in immature, quiescent cells nor LSCs. Strikingly, cytarabine-resistant pre-existing and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. Cytarabine residual cells exhibited increased fatty acid oxidation, upregulated CD36 expression and a HIGH OXPHOS gene signature predictive for treatment response in PDX and AML patients. HIGH OXPHOS but not LOW OXPHOS human AML cell lines were chemoresistant in vivo. Targeting mitochondrial protein synthesis, electron transfer, or fatty acid oxidation induced an energetic shift towards LOW OXPHOS and markedly enhanced anti-leukemic effects of cytarabine. Together, this study demonstrates that essential mitochondrial functions contribute to cytarabine resistance in AML and are a robust hallmark of cytarabine sensitivity and a promising therapeutic avenue to treat AML residual disease.

Summary Chromosomal translocations affecting Mixed Lineage Leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report development of highly potent and orally bioavailable small molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.

Key Points• Targeting of CD123 via CAR-engineered T cells results in rejection of human AML and myeloablation in mouse models.Many patients with acute myeloid leukemia (AML) are incurable with chemotherapy and may benefit from novel approaches. One such approach involves the transfer of T cells engineered to express chimeric antigen receptors (CARs) for a specific cell-surface antigen. This strategy depends upon preferential expression of the target on tumor cells. To date, the lack of AML-specific surface markers has impeded development of such CARbased approaches. CD123, the transmembrane a chain of the interleukin-3 receptor, is expressed in the majority of AML cells but is also expressed in many normal hematopoietic cells. Here, we show that CD123 is a good target for AML-directed CAR therapy, because its expression increases over time in vivo even in initially CD123 dim populations, and that human CD123-redirected T cells (CART123) eradicate primary AML in immunodeficient mice. CART123 also eradicated normal human myelopoiesis, a surprising finding because anti-CD123 antibody-based strategies have been reportedly well tolerated. Because AML is likely preceded by clonal evolution in "preleukemic" hematopoietic stem cells, our observations support CART123 as a viable AML therapy, suggest that CART123-based myeloablation may be used as a novel conditioning regimen for hematopoietic cell transplantation, and raise concerns for the use of CART123 without such a rescue strategy. (Blood. 2014;123(15):2343-2354

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