Page 76 - Haematologica - Vol. 105 n. 6 - June 2020

M.M. Majumder et al.
cells, we utilized a mass spectrometry-based quantitative proteomics approach to profile B cells (CD19+), T cells (CD3+) and monocytes (CD14+) derived from two healthy donors (Figure 5). We then employed CyTOF to compare the basal activity of nine proteins (in healthy and leukemic cell subsets) involved in MAPK, JAK-STAT, NF-κB and PI3K-mTOR signaling, which are commonly activated in many hematologic malignancies25-28 (Figure 6). By sample barcoding and subsequent pooling prior to antibody stain- ing, CyTOF allows for direct comparison of the phospho- rylation level of target proteins between multiple donors with high fidelity.19,20
Monocytes show higher expression of calprotectin (S100A8/S100A9), which is associated with dexamethasone resistance - By quantitative mass spectrometry-based pro- teomics, a total of 1,060 proteins were detected. Among these, 163, 131 and 13 proteins were only identified in CD3, CD14 and CD19 lysates, respectively (Figure 5A, Online Supplementary Table S7). The uniquely expressed proteins were associated with biological processes consis- tent with the functional differences between these cell types (Online Supplementary Figure S9). For instance, the proteome signature in monocytes was enriched in biolog- ical processes related to phagosome maturation (ATP6V0D1, CTSS, M6PR), autophagy (ATG3, LAMP2), PPAR-α/RARα activation (IL1β, p38 MAPK, GPD2, PLCG2), and STAT3 signaling (IGF2R, RAC1, p38 MAPK). Immunoglobulins (IGLL1, IGHA1) were identified in B- cell fractions. T cells expressed proteins related to T-cell receptor signaling (CD8A, CD247, LCK, ZAP70), granzyme signaling (GZMA, PRF1), and oxidative phos- phorylation (ATP5I, NDUFA8, NDUFB3, UQCRB). Besides observed differences in the abundance of proteins, variable expression in commonly detected proteins was noted (Figure 5B and C). Enzymes associated with scav- enging reactive oxygen species such as catalase (CAT) and glutathione peroxidase 1 (GPX1) were expressed at a sig- nificantly higher (P<0.001) level in monocytes (Figure 5B). In addition, monocytes exhibited elevated expression of isocitrate dehydrogenase 1 (IDH1), carboxylesterase 1 (CES1), and inflammatory protein calprotectin, a het- erodimer of two proteins S100A8/S100A9 that can medi- ate dexamethasone resistance in patients.29,30 We further compared the protein expression profiles for these cell subsets between healthy and four MM patients, and found an identical pattern of expression for CAT, GPX1 and S100A8/9 proteins (Figure 5D). While expression of 16 proteins differed between healthy and MM samples [false discovery rate (FDR) < 0.05], no significant differences were noted for CD14+ and CD3+ lysates (< 3 proteins).
Mapping shared signaling activities in healthy and leukemic hematopoietic cell subsets - NF-κB phosphorylation was detected in most cell types. Compared to other cell types, higher pNF-κB was detected in T/CD3 cells (Online Supplementary Figure S10). Significantly higher mTOR sig- naling, as measured by p4E-BP1 and pPLC-γ1, was observed in healthy CD34+CD38+ cells, monocytes, gran- ulocytes (neutrophils) and B cells (Figure 6A-C). These cell types also tended to have elevated sensitivity to omipalis- ib (PI3K/mTOR inhibitor) compared to other cell types in healthy or malignant samples (Figure 2B and Figure 7E). T cells lacking sensitivity to PI3K/mTOR inhibitors showed reduced mTOR signaling activity (Figure 6A-C).
CD34+CD38+ cells also exhibited high levels of ERK phos- phorylation (Figure 6A-C). ERK phosphorylation status, however, did not correlate to increased trametinib sensi- tivity in monocytes. An inverse relation between pSTAT3 levels and venetoclax sensitivity was observed among the different cell populations. Heightened levels of pSTAT3 were detected in monocytes and granulocytes, which lacked sensitivity to venetoclax (Figure 6A-D). In contrast, a lower level of pSTAT3 was observed in venetoclax sen- sitive B and NK cells. Two related but distinct cell types, CD3+CD4+ and CD3+CD8+ T cells, exhibited a difference in the level of pSTAT3 (Figure 6B) that might explain their subtle difference in sensitivity to venetoclax (Figure 6D). Comparison of signaling patterns detected in healthy PB or BM cells to corresponding leukemic cells expressing identical surface markers revealed remarkable similarity (Figure 6A-C), strengthening their association with cellular phenotypes. Furthermore, monitoring changes in signaling pattern for these proteins upon treatment with increasing concentrations (0 nM, 10 nM and 10 μM) of venetoclax in healthy PB (n=3), revealed that the directionality or mag- nitude of signaling changes for some of these proteins (i.e. pPLC-γ1 and pCREB) were also similar across these cell types (Online Supplementary Figure S11).
Innate drug sensitivities in cell subsets are retained in their malignant counterparts in different hematologic malignancies
To further confirm the similarity in drug responses between healthy and patient-derived cell subsets observed using the single cell assay, we compared ex vivo drug responses detected in bead-enriched healthy cells (CD3+, CD14+, CD19+, CD34+ and CD138+) to a cohort of 281 primary samples derived from multiple hematologic malignancies. For these analyses, we generated data using the CellTiter-Glo® viability assay. In agreement with non-selective effects detected on healthy cell types, bortezomib activity was detected in a wide range of hematologic malignancies (Figure 7A). The highest clo- farabine efficacy was observed in CD3+ T cells and in the T-cell prolymphocytic leukemia (T-PLL) patient subset (Figure 7B), which is reflective of the clinical success observed with other purine analogs (fludarabine or cladribine) in T-PLL. Reduced activity of the purine ana- log clofarabine was detected in both healthy and myelo- ma derived CD138+ cells. Although dexamethasone was found to be most effective in B-ALL and CLL, modest ex vivo effects were observed in other lymphocytic and plas- ma cell malignancies, including T-ALL, T-PLL, B-PLL, and MM (Figure 7C). Disease-specific acquisition of sensitiv- ity was also noted in a subset of AML patients, which was undetectable in healthy CD34+ (Figure 7C) or CD34+CD38+ (Figure 4B) cells. T-cell malignancies, simi- lar to healthy T cells, showed no response to omipalisib. Consistent with responses observed in healthy B cells, a higher response to venetoclax was detected in malignant B-cell types (Figure 7F). T-PLL samples also exhibited sensitivity to venetoclax, which has been tested more recently in two T-PLL patients with measurable clinical benefit.32 Venetoclax response agreed with navitoclax responses in B-cell diseases (Figure 7F). Increased sensi- tivity to navitoclax compared to venetoclax was detected in CML, T-ALL, and MM samples (Figure 7E and F). B- cell specific responses to midostaurin were detected in CLL and ALL samples (Figure 4C and Online
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