Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

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BackgroundFms-related tyrosine kinase 3 (FLT3) receptor serves as a prognostic marker and therapeutic target in acute myeloid leukemia (AML). Approximately one-third of AML patients carry mutation in FLT3, associated with unfavourable prognosis and high relapse rate. The multitargeted kinase inhibitor midostaurin (PKC412) in combination with standard chemotherapy (daunorubicin and cytarabine) was recently shown to increase overall survival of AML patients. For that reason, PKC412 has been approved for treatment of AML patients with FLT3-mutation. PKC412 synergizes with standard chemotherapy, but the mechanism involved is not fully understood and the risk of relapse is still highly problematic.MethodsBy utilizing the unique nature of mass cytometry for single cell multiparameter analysis, we have explored the proteomic effect and intracellular signaling response in individual leukemic cells with internal tandem duplication of FLT3 (FLT3-ITD) after midostaurin treatment in combination with daunorubicin or cytarabine.ResultsWe have identified a synergistic inhibition of intracellular signaling proteins after PKC412 treatment in combination with daunorubicin. In contrast, cytarabine antagonized phosphorylation inhibition of PKC412. Moreover, we found elevated levels of FLT3 surface expression after cytarabine treatment. Interestingly, the surface localization of FLT3 receptor increased in vivo on the blast cell population of two AML patients during day 3 of induction therapy (daunorubicin; once/day from day 1-3 and cytarabine; twice/day from day 1-7). We found FLT3 receptor expression to correlate with intracellular cytarabine (AraC) response. AML cell line cultured with AraC with or without PKC412 had an antagonizing phosphorylation inhibition of pAKT (p=0.042 and 0.0261, respectively) and pERK1/2 (0.0134 and 0.0096, respectively) in FLT3(high) compared to FLT3(low) expressing cell populations.ConclusionsOur study provides insights into how conventional chemotherapy affects protein phosphorylation of vital signaling proteins in human leukemia cells. The results presented here support further investigation of novel strategies to treat FLT3-mutated AML patients with PKC412 in combination with chemotherapy agents and the potential development of novel treatment strategies.

Dienone compounds with a 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore have been widely reported to show tumor cell selectivity. These compounds target the ubiquitin-proteasome system (UPS), known to be essential for the viability of tumor cells. The induction of oxidative stress, depletion of glutathione, and induction of high-molecular-weight (HMW) complexes have also been reported. We here examined the response of acute myeloid leukemia (AML) cells to the dienone compound VLX1570. AML cells have relatively high protein turnover rates and have also been reported to be sensitive to depletion of reduced glutathione. We found AML cells of diverse cytogenetic backgrounds to be sensitive to VLX1570, with drug exposure resulting in an accumulation of ubiquitin complexes, induction of ER stress, and the loss of cell viability in a dose-dependent manner. Caspase activation was observed but was not required for the loss of cell viability. Glutathione depletion was also observed but did not correlate to VLX1570 sensitivity. Formation of HMW complexes occurred at higher concentrations of VLX1570 than those required for the loss of cell viability and was not enhanced by glutathione depletion. To study the effect of VLX1570 we developed a zebrafish PDX model of AML and confirmed antigrowth activity in vivo. Our results show that VLX1570 induces UPS inhibition in AML cells and encourage further work in developing compounds useful for cancer therapeutics.

Vitamin D (Vit-D), an essential nutrient, interacts with different drugs including chemotherapeutic agents like busulphan, an alkylating agent used for conditioning prior to stem cell transplantation. The correlation between Vit-D plasma levels and busulphan clearance was investigated in an uncontrolled prospective study in patients and mice. Plasma 25(OH)D levels were measured and busulphan pharmacokinetics calculated in 81 patients. Adults received oral busulphan (n = 34) while children received busulphan orally (n = 19) or intravenously (n = 28). Patients received no Vit-D supplementation. To confirm our findings, pharmacokinetics after a single dose of busulphan (oral or intravenous) were evaluated in two groups of mice (n = 60) receiving high or standard-level Vit-D supplementation. Both busulphan clearance (P < 0.0001) and 25(OH)D levels (P = 0.0004) were significantly higher in adults compared to children. A significant negative correlation (P = 0.041) was found between busulphan clearance and 25(OH)D levels in children treated orally. No such correlation was observed in adults or in children receiving intravenous busulphan. In addition, no significant effect of Vit-D levels on busulphan pharmacokinetics in mice regardless of the administration route. In conclusion, 25(OH)D can affect oral busulphan pharmacokinetics in children and its level should be considered when personalizing oral busulphan treatment. Further studies are warranted to confirm the underlying mechanisms.

Treatments that include gemcitabine and carboplatin induce dose-limiting myelosuppression. The understanding of how human bone marrow is affected on a transcriptional level leading to the development of myelosuppression is required for the implementation of personalized treatments in the future. In this study, we treated human hematopoietic stem and progenitor cells (HSPCs) harvested from a patient with chronic myelogenous leukemia (CML) with gemcitabine/carboplatin. Thereafter, scRNA-seq was performed to distinguish transcriptional effects induced by gemcitabine/carboplatin. Gene expression was calculated and evaluated among cells within and between samples compared to untreated cells. Cell cycle analysis showed that the treatments effectively decrease cell proliferation, indicated by the proportion of cells in the G2M-phase dropping from 35% in untreated cells to 14.3% in treated cells. Clustering and t-SNE showed that cells within samples and between treated and untreated samples were affected differently. Enrichment analysis of differentially expressed genes showed that the treatments influence KEGG pathways and Gene Ontologies related to myeloid cell proliferation/differentiation, immune response, cancer, and the cell cycle. The present study shows the feasibility of using scRNA-seq and chemotherapy-treated HSPCs to find genes, pathways, and biological processes affected among and between treated and untreated cells. This indicates the possible gains of using single-cell toxicity studies for personalized medicine.

Since their introduction, tyrosine kinase inhibitors (TKIs, eg, imatinib, nilotinib, dasatinib, bosutinib, ponatinib) have revolutionized the treatment of chronic myeloid leukemia (CML). However, long-term treatment with TKIs is associated with serious adverse events including both bleeding and thromboembolism. Experimental studies have shown that TKIs can cause platelet dysfunction. Herein, we present the first side-by-side investigation comparing the effects of currently used TKIs on platelet function and thrombin generation when used in clinically relevant concentrations. A flow cytometry multiparameter protocol was used to study a range of significant platelet activation events (fibrinogen receptor activation, alpha granule, and lysosomal exocytosis, procoagulant membrane exposure, and mitochondrial permeability changes). In addition, thrombin generation was measured in the presence of TKIs to assess the effects on global hemostasis. Results show that dasatinib generally inhibited platelet function, while bosutinib, nilotinib, and ponatinib showed less consistent effects. In addition to these general trends for each TKI, we observed a large degree of interindividual variability in the effects of the different TKIs. Interindividual variation was also observed when blood from CML patients was studied ex vivo with whole blood platelet aggregometry, free oscillation rheometry (FOR), and flow cytometry. Based on the donor responses in the side-by-side TKI study, a TKI sensitivity map was developed. We propose that such a sensitivity map could potentially become a valuable tool to help in decision-making regarding the choice of suitable TKIs for a CML patient with a history of bleeding or atherothrombotic disease.

Changes in the immune system induced by tyrosine kinase inhibitors (TKI) have been shown to positively correlate with therapy responses in chronic myeloid leukemia (CML). However, only a few longitudinal studies exist and no randomized comparisons between two TKIs have been reported. Therefore, we prospectively analyzed the immune system of newly diagnosed CML patients treated with imatinib (n = 20) or bosutinib (n = 13), that participated in the randomized BFORE trial (NCT02130557). Comprehensive immunophenotyping, plasma protein profiling, and functional assays to determine activation levels of T and NK cells were performed at diagnosis, 3, and 12 months after therapy start. All results were correlated with clinical parameters such as Sokal risk and BCR-ABL load measured according to IS%. At diagnosis, low Sokal risk CML patients had a higher frequency of cytotoxic cells (CD8 + T and NK cells), increased cytotoxic potential of NK cells and lower frequency of naive and central memory CD4 + T cells. Further, soluble plasma protein profile divided patients into two distinct clusters with different disease burden at diagnosis. During treatment, BCR-ABL IS% correlated with immunological parameters such as plasma proteins, together with different memory subsets of CD4+ and CD8 + T cells. Interestingly, the proportion and cytotoxic potential of NK cells together with several soluble proteins increased during imatinib treatment. In contrast, no major immunological changes were observed during bosutinib treatment. In conclusion, imatinib and bosutinib were shown to have differential effects on the immune system in this randomized clinical trial. Increased number and function of NK cells were especially observed during imatinib therapy.

Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naive CD56(bright) NK cells had decreased relapse-free survival. In addition, the TNF-alpha/IFN-gamma cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.