Page 204 - Haematologica March 2020

H. Kuusanmäki et al.
Discussion
With FC-based drug testing we were able to simultane- ously measure drug sensitivities of different cell popula- tions in primary AML BM samples. Monocytic cells abun- dantly present in FAB M4/5 AML were markedly resistant to the Bcl-2 inhibitor venetoclax, while less differentiated blast cells in the same M4/5 samples or in M0/1/2 sam- ples were sensitive. Accordingly, the overall BM-MNC sensitivity to venetoclax was strongly influenced by FAB subtype. Our study shows that FC-based, phenotypic drug testing can improve the current understanding of ex vivo drug effects and may help to identify blast-specific treatments for AML patients.
Along with our previous studies, several other groups have evaluated ex vivo drug responses of Ficoll-enriched AML mononuclear cells using high-throughput CTG or MTS based cell viability assays.14,34–36 While these assays provide fast and robust readouts they fail to accurately measure blast specific drug responses. By using more accurate microscopy based screening, Snijder et al. have recently demonstrated that blast specific or relative blast fraction-based readouts increase predictive accuracy to treatment outcome.37 Similarly, Martinéz-Cuadrón et al. showed that a FC-based platform measuring blast specific effect in whole BM without MNC enrichment, predicted clinical response to induction therapy.25 We also showed earlier that in chronic myeloid leukemia, CD34-depleted cells (mature granulopoietic cells) were insensitive to BCR-ABL-1 inhibitors ex vivo whereas CD34+ progenitor cells showed good sensitivity.38 In accordance, we demon- strate here with a FC-based approach that blasts differ in their drug sensitivities in comparison to other cell popu- lations in the same AML samples. The highest blast-spe- cific efficacy was observed with venetoclax, whereas rux- olitinib and trametinib showed increased activity towards monocytic cells. Importantly, we demonstrate that in samples with a low blast count, the overall mononuclear cell fraction sensitivity does not correlate well with the blast-specific drug sensitivity.
Consistent with our results, earlier studies have shown that primary AML samples are sensitive to venetoclax ex vivo.15,39,40 Most of the studies have used mononuclear cell fractions to assess cell viability and to measure protein and gene expression levels. We observed that mononu- clear cells of M0/1 samples that mainly consisted of blasts, were sensitive to venetoclax compared to mononuclear cells of M4/5 samples when using a homog- enous CTG-based cell viability assay. Earlier, high ex vivo sensitivity to Bcl-2 inhibition has been associated with M3 AML in a study by Niu et al., whereas Pan et al. found no associations with FAB subtypes.39,40 Importantly, both study cohorts lacked comprehensive spectra of different subtypes, with none or only one M0/1 AML case. To sup- port our observation, mononuclear cells of M0/1 samples had a high BCL2/MCL1 gene expression ratio whereas M4/5 samples had a low ratio. Increased Bcl-2 protein expression has also been reported in M0/1 AML,41 and increased Mcl-1 expression in M4/5 AML26 of which the latter has been linked to elevated Mcl-1 expression in dif- ferentiating monocytes.42 Accordingly, we observed high MCL1 and BCL2A1 but low BCL2 expression in healthy monocytic and granulocytic cell populations.
By using a FC-based approach, we observed that sever- al M5 samples contained venetoclax-sensitive blasts and a
resistant monocytic cell fraction. This observation raises the question whether drug sensitivity profiling and gene/protein expression studies should focus on the immature blast cells and not the total MNC fraction espe- cially in in M4/5 samples and samples with low blast count. When we compared the FC measured blast-specif- ic venetoclax response between FAB subtypes, we observed a smaller but still significant difference between diagnosis M1 versus M5 subgroups. In clinical trials, NPM1, IDH1/2 and RUNX1 mutations have shown to be promising biomarkers for venetoclax+HMA treatment.7,43 Based on a study analyzing genotype and FAB subtype- specific patterns of 4,373 adult de novo AML cases,44 both IDH1/2 and RUNX1 mutations are enriched in M0/1/2 AML whereas NPM1 mutations are common in FAB M1/2/4/5 subtypes. Therefore, patient cohorts with mutated IDH1/2 or RUNX1 may be skewed to contain larger numbers of FAB M0/1/2 samples. To identify responders, it might be useful to evaluate the combined genetic and cell phenotype/FAB subtype information in a clinical setting.
With the FC method we also looked for effective com- binations, since an overall response rate of only 19% was observed with venetoclax monotherapy in patients with high-risk relapsed/refractory (R/R) AML.6 In our study, all venetoclax-resistant blasts showed sensitivity to MEK and/or JAK inhibitors suggesting that JAK/STAT and MAPK pathways play a major role in venetoclax resist- ance. We showed earlier that stromal cell secreted cytokines such as GM-CSF mediate resistance to veneto- clax, which can be counteracted by JAK inhibition.45 Moreover, the MAPK pathway plays a critical role in resistance through the proposed upregulation of MCL1.28 Both of these studies also demonstrated remarkable antileukemic activity in murine xenograft models when inhibiting JAK or MEK kinases together with Bcl-2. In agreement with the good synergism between ruxolitinib or trametinib with venetoclax observed here and in a recent study by the Beat AML study group,46 Kurtz et al. additionally showed that several different kinase inhibitors exhibited good synergism with venetoclax in AML samples.47 However, a recent clinical study with MEK inhibitor cobimetinib and venetoclax in R/R AML was closed due to limited clinical activity demonstrating that ex vivo drug screening results might not directly trans- late into a clinical setting.48
Inflammatory pathways are more active in M4/5 AML based on GSEA, consistent with the observed high sensi- tivity of monocytic cells to ruxolitinib and trametinib. Earlier studies have demonstrated that leukemic cells of patients with M4/5 AML produce IL1/IL613 and have a higher proliferative activity in cytokine-free medium.49 Thus, secreted cytokines and culturing conditions may have a big impact on the drug sensitivity profiles. While further investigation is warranted, results suggest that the JAK/STAT and MEK pathways are more active in differ- entiated monocytic cells as well as in venetoclax resistant blasts.
In summary, we show that ex vivo sensitivity of AML patient samples to venetoclax is associated with cell com- position. Furthermore, we demonstrate that FC-based drug screening could be implemented to identify effective targeted drugs and drug combinations against immature blasts, accelerating drug discovery and individualizing therapy for AML patients.
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haematologica | 2020; 105(3)