Signaling changes in the stem cell factor|[ndash]|AKT-S6 pathway in diagnostic AML samples are associated with disease relapse

Although current chemotherapy regimens are effective at eliminating the bulk of acute myeloid leukemia (AML) blasts, the disease relapses in the majority of patients.1 Whether relapse arises through the selection of a relatively rare subpopulation of cells present at diagnosis having specific characteristics that facilitate chemoresistance or through the induction of changes required to render them resistant to the chemotherapy is uncertain. Identification of the characteristics that confer chemoresistance, whether selected or induced, would allow for the implementation of additional therapies that target the lynchpin of resistance, thus improving disease outcome. This study evaluated whether single-cell network profiling (SCNP), a process that simultaneously measures post-translational protein modifications (for example, phosphorylation, acetylation and so on) in multiple signaling pathways within a single cell, could be used to functionally characterize intracellular pathways associated with in vivo AML chemotherapy resistance.2, 3 Specifically, we hypothesized that leukemic blasts at relapse have different signaling characteristics from those at diagnosis and that assessing intracellular signaling networks using the SCNP approach in longitudinally paired (that is, from the same patient) AML bone marrow (BM) samples at diagnosis and relapse time points may enable functional characterization of the mechanisms of chemoresistance in cell subpopulations that are dominant at relapse. The above hypothesis was tested by searching for resistance-associated signaling nodes in paired diagnosis/relapse samples from two AML patients and then examining the predictive value of the identified resistance-associated signaling nodes in an independent set of diagnostic AML samples collected from patients that had a complete disease response4 to standard induction chemotherapy and for which clinical annotation about disease relapse was available. Clinical characteristics of the two AML patients are shown in Table 1. Both patients received high-dose cytarabine-based induction chemotherapy with complete disease response followed by relapse within 1 year. Cytogenetic analysis revealed prognostically unfavorable translocations of AML1-EVI1 and DEK-NUP214 [t(6;9)] in patients 1 and 2, respectively. In addition, patient 2 had FMS-like tyrosine kinase 3 receptor internal tandem duplications (FLT3-ITD)-positive leukemia, a known poor prognostic marker for relapse risk and overall survival and associated with the DEK-NUP214 translocation in the majority of cases.5 Healthy control BM mononuclear cells (N=2) were derived from young healthy male volunteers (age=18 and 20 years, respectively). The cryopreserved BM samples from the above two AML patients were processed (thawed, modulated, fixed, permeabilized and incubated with antibodies to both surface and intracellular proteins)2, 3, 6 to assess whether specific cell subpopulations identified using cell surface phenotypes and/or signaling profiles were present in the relapsed sample in a greater percentage than observed in the corresponding diagnostic sample (Figure 1). Next, in an independent and larger group of diagnostic patient samples, blasts with the previously identified cell profiles were examined for their association with disease relapse (Figure 1). The two longitudinally paired samples were first compared for expression of conventional surface markers used to define myeloblast maturity as shown in Figure 2a.7 Cell subpopulations based on these characteristics of myeloblast maturity were not informative of relapse risk for either patient sample (Figure 2b). Examination of intracellular signaling profiles revealed functionally distinct cell subsets in the otherwise phenotypically similar relapse and diagnosis samples (Figure 3 and Supplementary Figure 1). Specifically, when the relapse samples from patient 1 and patient 2 were modulated using stem cell factor (SCF), both p-Akt and p-S6 were induced in 3.2 and 31.7% of cells, respectively (Figure 3). A similar finding of an increased percentage of myeloblast subpopulations defined by intracellular signaling profiles in relapse versus diagnosis samples was observed when FLT3L (inducing p-S6 and p-Akt, Figure 3) and interleukin-27 or granulocyte colony-stimulating factor (inducing p-signal transducer and activator of transcription (STAT)3 and p-STAT5) were used as modulators (Supplementary Figure 1). To investigate whether similar cells were present at the time of diagnosis, which would support the concept of selection, or absent, supporting the idea of an induced change, we looked for the presence of cells with similar functional responses to SCF, FLT3L, interleukin-27 and granulocyte colony-stimulating factor in the corresponding diagnosis samples. While no interleukin-27-responsive subpopulation was identified, SCF-, FLT3L- and granulocyte colony-stimulating factor-responsive cells were observed in the diagnostic AML BM samples (Figure 3 and Supplementary Figure 1), although in a lower percentage (~1%). SCF-responsive cells, defined by the SCF:p-Akt/p-S6 signaling profile, were phenotypically diverse in the relapse AML samples compared with SCF-responsive cells in healthy donor BM samples (Figure 4). In healthy BM samples, the SCF: p-Akt/p-S6 profile existed specifically in a subset of progenitor cells with the CD34+, CD33−, CD11b− phenotype (Figure 4). After identifying the resistance-associated signaling nodes in the relapsed samples, we hypothesized that detection of the same nodes in the diagnostic samples might be predictive of poor outcome (early relapse). We first applied the SCF:p-Akt/p-S6 gating scheme (as defined in Supplementary Figure 2) to an independent set of diagnostic AML samples. The clinical characteristics of these 47 patients are shown in Table 2. All patients received high-dose cytarabine-based induction chemotherapy with disease response of complete remission. Of these, 27 experienced disease relapse (complete response followed by relapse, CR Rel), while 20 remained in complete continuous remission for two or more years. Patients from whom this independent sample set was obtained were young (41/47; <60) and a high proportion (20/47) had French-American-British classification system M2 AML. In seven diagnostic AML samples, a subset of leukemic blast cells that responded to SCF modulation by phosphorylation of p-Akt and p-S6 were observed (Table 2, Supplementary Table S1). Of those seven, six patients experienced disease relapse within 2 years (P=0.21, Fisher's exact test) from remission, while the seventh patient had a complete remission lasting more than 2 years; interestingly, this AML sample had favorable cytogenetics, t(8;21) (Figure 5 and Supplementary Table S1). Of note, all of the patients with this SCF-responsive profile were less than 60 years old and, with the exception mentioned above, they all had intermediate- or high-risk cytogenetics; six out of seven also had an early myeloid FAB classification of M1 or M2. Also of note, the occurrence of the SCF:pAkt/pS6 subpopulation was independent of the presence of FLT3-ITD: only one of the six samples was positive for FLT3-ITD mutation. Importantly, the predictive value of the combination of FLT3-ITD and SCF:p-Akt/p-S6 for disease relapse was greater than that of either biomarker individually (P=0.03, Fisher's exact test, Table 3). We next examined whether expression of c-Kit, the receptor for SCF, could function as a surrogate marker for the SCF:p-Akt/p-S6 phenotype. Although only samples that expressed c-Kit were able to respond to SCF, no association between c-Kit expression levels and likelihood of leukemia relapse (Figure 6a) was observed, suggesting that c-Kit expression is a necessary but not sufficient condition for SCF-induced intracellular signaling. In line with this observation, the removal of non-c-Kit-expressing samples improved relapse prediction (Figure 6b). Furthermore, when blast cells from an AML sample were simultaneously examined for c-Kit and the downstream signaling marker p-Akt, intra-patient heterogeneity in c-Kit expression and response to SCF within c-Kit-expressing cells were observed (Figure 6c). We also examined whether FLT3L:p-Akt/p-S6, granulocyte colony-stimulating factor:p-STAT3/5 or interleukin-27:p-STAT3/5 signaling nodes predicted poor outcome in the same independent set of diagnostic AML samples. Unlike SCF:p-Akt/p-S6 gate, no association was found with disease relapse (Supplementary Table S2). Relapse due to chemoresistant residual disease is a major cause of death in both adult and pediatric patients with AML, and aberrant signal transduction within pathways that control cell proliferation and survival is thought to have an important role in secondary chemoresistance. In this study, we used SCNP as a strategy to identify specific signaling pathway profiles associated with in vivo chemoresistance using paired diagnosis and relapse samples. Although it was performed on a limited number of paired AML samples, our study provides unique insights into the nature of AML secondary chemoresistance in rare cell populations, identifying a functionally characterized cell subset associated with likelihood of early relapse when the assay was applied in a separate patient cohort. A subset of leukemia cells with enhanced activity within the phosphoinositide 3-kinase/Akt cascade (SCF:p-Akt/p-S6) was found to be commonly expanded in the two leukemia samples collected at relapse. Importantly, the presence of cell subpopulations expressing this same signaling profile at diagnosis was associated with disease relapse after complete response to induction chemotherapy in an independent sample set of AML diagnostic samples. Although the SCF:p-Akt/p-S6 profile was not present in all patients with relapsed disease, all but one patient with a diagnostic sample containing a subpopulation of 3% SCF:p-Akt/p-S6 cells relapsed within 2 years of remission. These data support the marked biological heterogeneity at the basis of AML secondary chemoresistance and lend merit to the approach of studying signaling profiles in functionally distinct subpopulations in longitudinally collected AML samples before and after therapy to identify poor-prognostic cell populations. Currently there are no measures to indicate why patients with similar clinically appearing disease have different responses to therapy, with some remaining disease free while others undergo disease relapse and ultimately succumb. SCNP permits an accurate characterization of each individual's leukemia signaling pathway phenotype and biological heterogeneity, allowing for a more efficient delineation of the normality or pathology of leukemic subpopulations. This study shows that leukemic cell populations differ quantitatively and qualitatively before and after in-vivo therapeutic pressure in AML, and that SCNP offers a novel approach to identify chemotherapy-resistant subpopulations that may predispose patients to disease relapse. Dr Cohen, Dr Woronicz and Dr Fantl were employees of Nodality during the time the research was conducted. Todd Covey, Dr Putta, Dr Gayko and Dr Cesano were employees of Nodality during the time the research was conducted and are presently employed by Nodality. Dr Kornblau declares no conflict of interest. Supplementary Information accompanies the paper on Blood Cancer Journal website