Early response evaluation by single cell signaling profiling in acute myeloid leukemia.

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phospho-ERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.

Implementing a Functional Precision Medicine Tumor Board for Acute Myeloid Leukemia.

We generated ex vivo drug-response and multiomics profiling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identification of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profiling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes significant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275.

Sex disparity in acute myeloid leukaemia with FLT3 internal tandem duplication mutations: implications for prognosis.

Incidence, molecular presentation and outcome of acute myeloid leukaemia (AML) are influenced by sex, but little attention has been directed at untangling sex-related molecular and phenotypic differences between female and male patients. While increased incidence and poor risk are generally associated with a male phenotype, the poor prognostic FLT3 internal tandem duplication (FLT3-ITD) mutation and co-mutations with NPM1 and DNMT3A are overrepresented in female AML. Here, we have investigated the relationship between sex and FLT3-ITD mutation status by comparing clinical data, mutational profiles, gene expression and ex vivo drug sensitivity in four cohorts: Beat AML, LAML-TCGA and two independent HOVON/SAKK cohorts, comprising 1755 AML patients in total. We found prevalent sex-associated molecular differences. Co-occurrence of FLT3-ITD, NPM1 and DNMT3A mutations was overrepresented in females, while males with FLT3-ITDs were characterized by additional mutations in RNA splicing and epigenetic modifier genes. We observed diverging expression of multiple leukaemia-associated genes as well as discrepant ex vivo drug responses, suggestive of discrete functional properties. Importantly, significant prognostication was observed only in female FLT3-ITD-mutated AML. Thus, we suggest optimization of FLT3-ITD mutation status as a clinical tool in a sex-adjusted manner and hypothesize that prognostication, prediction and development of therapeutic strategies in AML could be improved by including sex-specific considerations.

FLT3-ITD mutations in acute myeloid leukaemia - molecular characteristics, distribution and numerical variation.

Recurrent somatic internal tandem duplications (ITD) in the FMS-like tyrosine kinase 3 (FLT3) gene characterise approximately one third of patients with acute myeloid leukaemia (AML), and FLT3-ITD mutation status guides risk-adapted treatment strategies. The aim of this work was to characterise FLT3-ITD variant distribution in relation to molecular and clinical features, and overall survival in adult AML patients. We performed two parallel retrospective cohort studies investigating FLT3-ITD length and expression by cDNA fragment analysis, followed by Sanger sequencing in a subset of samples. In the two cohorts, a total of 139 and 172 mutant alleles were identified in 111 and 123 patients, respectively, with 22% and 28% of patients presenting with more than one mutated allele. Further, 15% and 32% of samples had a FLT3-ITD total variant allele frequency (VAF) < 0.3, while 24% and 16% had a total VAF ≥ 0.7. Most of the assessed clinical features did not significantly correlate to FLT3-ITD numerical variation nor VAF. Low VAF was, however, associated with lower white blood cell count, while increasing VAF correlated with inferior overall survival in one of the cohorts. In the other cohort, ITD length above 50 bp was identified to correlate with inferior overall survival. Our report corroborates the poor prognostic association with high FLT3-ITD disease burden, as well as extensive inter- and intrapatient heterogeneity in the molecular features of FLT3-ITD. We suggest that future use of FLT3-targeted therapy could be accompanied with thorough molecular diagnostics and follow-up to better predict optimal therapy responders.

Single Cell Detection of the p53 Protein by Mass Cytometry.

PURPOSE: The p53 protein and its post-translational modifications are distinctly expressed in various normal cell types and malignant cells and are usually detected by immunohistochemistry and flow cytometry in contemporary diagnostics. Here, we describe an approach for simultaneous multiparameter detection of p53, its post-translational modifications and p53 pathway-related signaling proteins in single cells using mass cytometry. METHOD: We conjugated p53-specific antibodies to metal tags for detection by mass cytometry, allowing the detection of proteins and their post-translational modifications in single cells. We provide an overview of the antibody validation process using relevant biological controls, including cell lines treated in vitro with a stimulus (irradiation) known to induce changes in the expression level of p53. Finally, we present the potential of the method through investigation of primary samples from leukemia patients with distinct TP53 mutational status. RESULTS: The p53 protein can be detected in cell lines and in primary samples by mass cytometry. By combining antibodies for p53-related signaling proteins with a surface marker panel, we show that mass cytometry can be used to decipher the single cell p53 signaling pathway in heterogeneous patient samples. CONCLUSION: Single cell profiling by mass cytometry allows the investigation of the p53 functionality through examination of relevant downstream signaling proteins in normal and malignant cells. Our work illustrates a novel approach for single cell profiling of p53.

AXL Targeting Abrogates Autophagic Flux and Induces Immunogenic Cell Death in Drug-Resistant Cancer Cells.

INTRODUCTION: Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC. METHODS: We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC. RESULTS: We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018). CONCLUSION: Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

Multi-parametric single cell evaluation defines distinct drug responses in healthy hematologic cells that are retained in corresponding malignant cell types.

Innate drug sensitivity in healthy cells aids identification of lineage specific anti-cancer therapies and reveals off-target effects. To characterize the diversity in drug responses in the major hematopoietic cell types, we simultaneously assessed their sensitivity to 71 small molecules utilizing a multi-parametric flow cytometry assay and mapped their proteomic and basal signaling profiles. Unsupervised hierarchical clustering identified distinct drug responses in healthy cell subsets based on their cellular lineage. Compared to other cell types, CD19+/B and CD56+/NK cells were more sensitive to dexamethasone, venetoclax and midostaurin, while monocytes were more sensitive to trametinib. Venetoclax exhibited dose-dependent cell selectivity that inversely correlated to STAT3 phosphorylation. Lineage specific effect of midostaurin was similarly detected in CD19+/B cells from healthy, acute myeloid leukemia and chronic lymphocytic leukemia samples. Comparison of drug responses in healthy and neoplastic cells showed that healthy cell responses are predictive of the corresponding malignant cell response. Taken together, understanding drug sensitivity in the healthy cell-of-origin provides opportunities to obtain a new level of therapy precision and avoid off-target toxicity.

Modulation of phospho-proteins by interferon-alpha and valproic acid in acute myeloid leukemia.

PURPOSE: Valproic acid (VPA) is suggested to be therapeutically beneficial in combination with interferon-alpha (IFNα) in various cancers. Therefore, we examined IFNα and VPA alone and in combinations in selected AML models, examining immune regulators and intracellular signaling mechanisms involved in phospho-proteomics. METHODS: The anti-leukemic effects of IFNα and VPA were examined in vitro and in vivo. We mapped the in vitro phosphoprotein modulation by IFNα-2b and human IFNα-Le in MOLM-13 cells by IMAC/2D DIGE/MS analysis and phospho-flow cytometry, and in primary healthy and AML patient-derived PBMCs by CyTOF. In vivo, IFNα-Le and VPA efficacy were investigated in the immunodeficient NOD/Scid IL2γ-/- MOLM-13Luc+ mouse model and the syngeneic immunocompetent BNML rat model. RESULTS: IFNα-2b and IFNα-Le differed in the modulation of phospho-proteins involved in protein folding, cell stress, cell death and p-STAT6 Y641, whereas VPA and IFNα-Le shared signaling pathways involving phosphorylation of Akt (T308), ERK1/2 (T202/T204), p38 (T180/Y182), and p53 (S15). Both IFNα compounds induced apoptosis synergistically with VPA in vitro. However, in vivo, VPA monotherapy increased survival, but no benefit was observed by IFNα-Le treatment. CyTOF analysis of primary human PBMCs indicated that lack of immune-cell activation could be a reason for the absence of response to IFNα in the animal models investigated. CONCLUSIONS: IFNα-2b and IFNα-Le showed potent and synergistic anti-leukemic effects with VPA in vitro but not in leukemic mouse and rat models in vivo. The absence of IFNα immune activation in lymphocyte subsets may potentially explain the limited in vivo anti-leukemic effect of IFNα-monotherapy in AML.

Titrating Complex Mass Cytometry Panels.

We describe here a simple and efficient antibody titration approach for cell-surface markers and intracellular cell signaling targets for mass cytometry. The iterative approach builds upon a well-characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. Healthy peripheral blood and bone marrow cells are stained with a pre-optimized "backbone" antibody panel in addition to the progressively diluted (titrated) antibodies. Clustering based on the backbone panel enables the titration of each antibody against a rich hematopoietic background and assures that nonspecific binding and signal spillover can be quantified accurately. Using a slightly expanded backbone panel, antibodies quantifying changes in transcription factors and phosphorylated antigens are titrated on ex vivo stimulated cells to optimize sensitivity and evaluate baseline expression. Based on this information, complex panels of antibodies can be thoroughly optimized for use on healthy whole blood and bone marrow and are easily adaptable to the investigation of samples from for example clinical studies. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Cellular context-mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis.

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell-cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis.

Akt1 activity regulates vessel maturation in a tissue engineering model of angiogenesis.

Akt kinase is a central signal transduction node that integrates extracellular cues that regulate cell migratory, proliferative, and morphological functions during angiogenesis. However, how Akt activity is modulated and contributes to subsequent vessel maturation is unclear. In this study we investigated the role of Akt1 in vessel maturation using human dermal microvascular endothelial cells (HDMVECs) expressing constitutively active and hemiphosphorylated Akt1 epi-alleles with graded kinase activity. HDMVECs expressing Akt1 epi-alleles were analyzed in vivo in a tissue engineering setting using a model of angiogenesis comprising cell-seeded poly-L-lactic acid scaffolds implanted subcutaneously into NOD/SCID murine hosts. The resultant intraimplant microvasculature was quantified for vascular parameters, including vessel diameter, perfusion, vascular density, and pericyte coverage. We found that constitutive Akt1 kinase activity in implanted HDMVECs correlated with loss of neovasculature function. Further, we found that the presence of coimplanted vascular smooth muscle cells (vSMCs) in the implants failed to promote blood vessel growth and maturation in a graded, Akt1 kinase activity-dependent manner. These results indicate that constitutive Akt1 activity disrupts the normal blood vessel growth and maturation. Therefore, we suggest that a downregulation of Akt1 activity is necessary for vSMC-induced maturation of newly formed blood vessels to occur.

Efficient in vivo vascularization of tissue-engineering scaffolds.

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly-L-lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue-engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non-random and displayed a preferential perivascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.