The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed “minimal residual disease”. The phenomenon of disease persistence suggests that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of leukemic stem cells (LSCs). Although other markers of a primitive CML LSC population have been identified in the preclinical setting, only CD26 appears to offer clinical utility. Here we demonstrate consistent and selective expression of CD93 on a lin⁻CD34⁺CD38⁻CD90⁺CML LSC population and show in vitro and in vivo data to suggest increased stem cell characteristics, as well as robust engraftment in patient-derived xenograft models in comparison with a CD93⁻CML stem/progenitor cell population, which fails to engraft. Through bulk and single-cell analyses of selected stem cell and cell survival-specific genes, we confirmed the quiescent character and demonstrate their persistence in a population of CML patient samples who demonstrate molecular relapse on TKI withdrawal. Taken together, our results identify that CD93 is consistently and selectively expressed on a lin⁻CD34⁺CD38⁻CD90⁺CML LSC population with stem cell characteristics and may be an important indicator in determining poor TKI responders.

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and antitumor efficacy compared with the high-affinity (FMC63-based) CAR used in tisagenlecleucel, in preclinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a phase 1 clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T cells, we performed a systematic in vitro characterization of the transcriptomic (RNA sequencing) and protein (cytometry by time of flight) changes occurring in T cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared with FMC63 CAR T cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B cells present in the manufacture.

Trib2 expression in granulocyte-monocyte progenitors drives a highly drug resistant acute myeloid leukaemia linked to elevated Bcl2 Trib2 expression in granulocyte-monocyte progenitors drives a highly drug resistant acute myeloid leukaemia linked to elevated Bcl2

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2 Tyrosine kinase inhibitors (TKI) have revolutionised the treatment of CML. However, TKI do not eliminate the leukaemia stem cells (LSC), which can reinitiate the disease. Thus, finding new therapeutic targets in CML LSC is key to finding a curative treatment. Using microarray datasets, we defined a list of 227 genes which were differentially expressed in CML LSC compared to healthy controls but were not affected by TKI in vitro . Two of them, CD33 and PPIF , are targeted by gemtuzumab-ozogamicin and cyclosporin A, respectively. We treated CML and control CD34 ⁺ cells with either drug with or without imatinib to investigate the therapeutic potential of the TKI-independent gene expression programme. Cyclosporine A in combination with imatinib reduced the number of CML CFC compared with non-CML controls, but only at supra-therapeutic concentrations. Gemtuzumab-ozogamicin showed a EC ₅₀ of 146ng/mL, below the plasma peak concentration of 630ng/mL observed in AML patients and below the EC ₅₀ of 3247ng/mL observed in non-CML cells. Interestingly, gemtuzumab-ozogamicin seems to promote cell cycle progression in CML CD34 ⁺ cells and demonstrated activation of the RUNX1 pathway in a RNAseq experiment. This suggests that targeting the TKI-independent genes in CML LSC could be exploited for the development of new therapies in CML. 1 Simple summary: Chronic myeloid leukaemia (CML) is initiated by a group of cancer cells called leukaemia stem cells (LSC). These LSC can survive current tyrosine kinase inhibitor (TKI) treatments and upon treatment withdrawal, are able to re-initiate the disease. Thus, eradicating the LSC would likely cure CML. In this study, we have identified a number of genes which expression is different between LSC and their healthy counterparts (haematopoietic stem cells) but are not affected by TKI treatment. We hypothesised that these genes may be potential therapeutic targets against LSC and used two different drugs, gemtuzumab-ozogamicin and cyclosporine A, to treat CML in vitro . We found that both drugs have a stronger effect on CML cells than in healthy cells. Therefore, we propose that the list of genes we identified could represent a novel source of therapeutic targets against CML.