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Venetoclax response and maturation stage in AML
each assay to DSS (a drug sensitivity metric based on area under the dose-response curve, higher DSS indicates high- er sensitivity).16 We observed a strong correlation between CTG and FC viability derived DSS when all live CD45+ leukocytes were used as the FC readout (R=0.64, P<0.0001, Figure 2A), and when the blast-specific drug responses were exclusively taken as the FC readout from samples with blast counts over 50% (R=0.75, P<0.0001, Figure 2B). However, we observed poor correlation between the FC and CTG results in a sample cohort with blast counts below 50% (R=0.24, P=0.05, Figure 2C). The most prominent differences were seen in the response to trametinib and venetoclax (Online Supplementary Figure S3). The poor correlation was partly due to highly differ- ent drug sensitivities of the non-blast cell populations compared to blasts as demonstrated in two samples with low blast counts (Figure 2D-G). Our data shows that AML BM subpopulations have heterogeneous drug responses that confound the assessment of blast specific drug sensi- tivities when using homogenous cell viability assays in unsorted BM-MNC samples.
Ex vivo drug screening predicts induction therapy response
Next, we evaluated whether incomplete BM blast clear- ance at day 14 and day 28 after induction treatment was associated with decreased ex vivo drug sensitivity. We evaluated BM samples from 15 patients collected prior to anthracycline+cytarabine induction chemotherapy. Amongst these patients, five had >10% blast cells at day 14 and/or day 28 and were defined as chemoresistant as described in the Online Supplementary Table S1. Additionally, we included samples from two patients resistant to induction (collected at the time of resistant dis- ease) in the chemoresistant group. A combined DSS of cytarabine and idarubicin showed significantly lower val- ues for the resistant patients both with FC and (P<0.05, 2H) and CTG (P<0.01, Figure 2I). Furthermore, we observed a significant difference between responders and non-responders when blast-specific idarubicin response was measured with FC (P<0.05, Figure 2H) or total sample sensitivity was measured with CTG (P<0.05, Figure 2I). These results are in line with a recent study demonstrating that a similar FC-based platform can predict induction therapy response in a larger AML cohort.25
Blasts are highly sensitive to Bcl-2 inhibition whereas monocytes and granulocytes are resistant
Using the FC approach, we were able to evaluate blast- specific drug responses and compare them to other cell types within the same or between samples. Amongst the seven tested drugs, venetoclax (IC50=3.0nM) and idaru- bicin (IC50=28.7nM) showed the highest toxicity against blasts (Table 1). However, between these two drugs vene- toclax showed the most selective efficacy against blasts when compared to other cell populations and healthy CD34+ cells (Figure 3A, IC50 values in the Online Supplementary Figure S4). Moreover, venetoclax was also effective against CD34+CD38– cells, which suggests activ- ity against leukemic stem cells (Online Supplementary Figure 5). Compared to blasts, monocytic cells (CD14+) were highly resistant to Bcl-2 inhibition (P<0.001, Mann-Whitney U test), but sensitive to MEK and JAK inhibition (P<0.001, Figure 3A). The phenomenon was clearly observed in samples from patients diagnosed with
acute monocytic leukemia (M5) that contained substantial fractions of both cell types (Figure 3B-C).
Overall BM AML sample sensitivity to venetoclax is associated with FAB subtype
To follow-up on our findings, we hypothesized that AML samples with a high monocytic cell content should have a distinct drug response profile when overall BM- MNC sensitivity is measured with the CTG assay. We re- analyzed our earlier published CTG-based drug sensitivity data of 37 AML samples comprised of FAB M1, M2, M4 and M5 samples that were screened with 296 compounds.14,15 Amongst the 296 compounds, venetoclax showed the largest drug sensitivity difference between M1 and M5 AML (P<0.001, Online Supplementary Table S4, Figure 4A). Similarly, the CTG-based sensitivity of the AML sample cohort studied here showed a gradual decrease in venetoclax sensitivity from M0 towards M5 subtype (Figure 4B). When we limited our FC analysis to diagnostic samples, a significant but smaller difference in blast-specific venetoclax sensitivity was also associated with FAB subtype (P<0.05, Figure 4C). This significance was not observed when we also included relapse and chemorefractory samples in the analysis (Figure 4D) large- ly due to a high number of chemorefractory M1/2 samples in our cohort that were more resistant to venetoclax (P<0.001, Figure 4D-E). Taken together, monocytic cells blur the high blast specific venetoclax effect in Ficoll- enriched M4/5 samples when measured with CTG but FAB subtype still has a significant effect on venetoclax response in blasts in our diagnosis AML sample cohort.
FAB subtype is associated with BCL2 and MCL1 gene expression
Anti-apoptotic Mcl-1 and Bcl-2 are considered the most important pro-survival factors in AML.26,27 Furthermore, their expression and phosphorylation has been shown to be regulated through the Ras/Raf/MEK/ERK, PI3K/PTEN/AKT and JAK/STAT signal transduction path- ways in different leukemias.28–31 To study whether the expression of BCL2 family members and activity of signal transduction pathways is associated with FAB subtypes, we analyzed gene expression data of MNC of diagnosis AML samples using publicly available microarray and RNA-seq data. BCL2 was highly expressed in M0/1 AML and gradually decreased towards M5 samples and healthy monocytes (Figure 5A, Online Supplementary Figure S6). Notably, MCL1 showed an opposite trend in expression and was most highly expressed in healthy monocytes (Figure 5A). We also detected higher expression of BCL2A1, BCL2L11 (BIM), BID and JAK2 in M4/5 AML. A more detailed analysis of the healthy myeloid compart- ment revealed that BCL2 family expression is highly dependent on differentiation stage, which likely also influ- ences the expression patterns seen between the different FAB subtypes (Figure 5B). Interestingly, high BCL2 and low MCL1 expression was also observed in FAB M3 AML and their healthy counterparts, colony forming unit (CFU) granulocytes (Figure 5A-B). High BCL2/MCL1 expression ratio in CFU granulocytes might explain the neutropenia seen in venetoclax treated patients.
Next, we investigated whether common cytogenetic abnormalities (RUNX1-RUNX1T1, CBFB-MYH11, MLL, PML-RARA) or mutations (FLT3, NPM1, RUNX1, CEBPA) explain some of the variations we observed in MCL1,
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