Capitalizing on paradoxical activation of the mitogen-activated protein kinase pathway for treatment of Imatinib-resistant mast cell leukemia.

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation." In the present work we re-analyzed the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KIT V560G, D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSAKIT D816V and in KIT D816V hematopoietic progenitors obtained from patient-derived induced pluripotent stem cells (iPS cells) and systemic mastocytosis patient samples. In conclusion, mutated KIT-driven Imatinib resistance and possible TKI-induced paradoxical activation can be efficiently overcome by a low concentration Ponatinib and Trametinib co-treatment, potentially reducing the negative side effects associated with MCL therapy.

Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced, AML remains a therapeutic challenge with insufficient cure rates. In the last years, immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced, which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However, the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here we report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression, unimpeded proliferation, and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore, CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects.

CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity.

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33- subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33- NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33- NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.