Yury Goltsev

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

Dysregulated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F-negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34(+) early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

Unipotent Megakaryopoietic Pathway Bridging Hematopoietic Stem Cells and Mature Megakaryocytes

Recent identification of platelet/megakaryocyte‐biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIbα) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41+ cells in the lineage Sca1+cKit+ (LSK) fraction (subCD41+LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41+LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3+CD41−LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41+LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO‐mediated phosphorylation. On increased demand of platelet production after 5‐FU treatment, a part of CD41+LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41+CD42b+LSK are straightforward progenies of megakaryocytes/platelet‐biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis. Stem Cells 2015;33:2196–2207

Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging

Summary A highly multiplexed cytometric imaging approach, termed co-detection by indexing (CODEX), is used here to create multiplexed datasets of normal and lupus (MRL/lpr) murine spleens. CODEX iteratively visualizes antibody binding events using DNA barcodes, fluorescent dNTP analogs, and an in situ polymerization-based indexing procedure. An algorithmic pipeline for single-cell antigen quantification in tightly packed tissues was developed and used to overlay well-known morphological features with de novo characterization of lymphoid tissue architecture at a single-cell and cellular neighborhood levels. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell-interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed spatial cytometry demonstrated here allows for quantitative systemic characterization of tissue architecture in normal and clinically aberrant samples.

Abstract A089: Multiparametric immunofluorescence analysis of the tumor microenvironment using CODEX

The tumor microenvironment plays a critical role in cancer progression and has implications for the efficacy of various cancer immunotherapy treatment options. Immune infiltrates within the tumor microenvironment can correlate with both positive and negative outcomes, depending upon the both the type of cancer as well as infiltrating immune cell(s). These analyses are typically performed using standard immunofluorescence and immunohistochemistry assays where no more than four simultaneous parameters can be visualized on the same tissue. Unfortunately, these tools cannot fully characterize the complexity of the tumor microenvironment due to the inherent limitations of fluorophore spectral overlap. In order to identify each type of immune and tumor cell within a single tissue, at least 40 parameters need to be measured simultaneously. We have developed a multiparametric immunofluorescence technology, entitled CODEX (Co-Detection by IndEXing), which utilizes unique DNA tags as a means of iteratively measuring more than 40 parameters within the same tissue. More than 40 human antibodies have been validated using this approach, including numerous immune markers, checkpoint ligands, tumor markers and cellular activity markers. We are currently analyzing tissue sample from patients with lung cancer. By measuring nearly 50 simultaneous markers within the same tissue, CODEX has the potential to greatly enhance our knowledge of the tumor microenvironment and more accurately define immune infiltrates at the single-cell level. Citation Format: Julia Kennedy-Darling, Garry P. Nolan, Yury Goltsev, Nikolay Samusik. Multiparametric immunofluorescence analysis of the tumor microenvironment using CODEX [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A089.

Abstract PR14: Automatic identification of cell niches and immune interactions important for clinical outcomes using multiparameter imaging and deep neural networks

Multiparameter cytometry, for example with CyTOF, has enabled the interrogation of immune phenotypes in unprecedented detail in many clinical contexts. But cytometry is incapable of answering a question of critical importance to many tissue context studies, and especially understanding how local interactions between tumor cells and immune cells correlate to clinical outcomes. This becomes especially relevant to understanding the subtleties of how different immunotherapeutic approaches operate in vivo. We recently developed a multiparameter immunofluorescence technique, termed CODEX, which allows the capture of spatial information for protein and RNA expression in tissue sections. This spatial information enables us to establish not only cell-types according to traditional phenotypic surface marker expression, but also to potentially surmise specific tissue states driving clinical responses. To make sense of the high-dimensional data afforded by CODEX, we apply here state-of-the-art deep neural networks (DNNs). These networks, which have achieved superhuman classification accuracy in many diverse domains, automatically identify cells, cell niches and regions (at multiple scales) that are capable of distinguishing healthy and diseased samples. This is done in an unbiased way, with only ‘healthy’ vs. ‘disease’ labels as additional input alongside the imaging data. We first train DNNs to successfully classify multiparameter tissue images from independent replicates across conditions. Having achieved a high accuracy of classification, we set the network output to highlight cells and regions deemed to be most relevant to classify each condition. Applying this methodology to healthy and mrl (lupus) spleens stained for 30 markers, our neural network is able to successfully identify a not previously observed enrichment of cell confluences (niches) consisting of CD8 T-cells and conventional dendritic cells enriched in MRL samples, as well as other novel niches completely unpredicted by prior knowledge. Our DNN enables the systematic and unbiased discovery of specific immune interactions in any tissue type. Applying our technique to the analysis of samples from immunotherapy recipients could enable the discovery of key factors in the tumor microenvironment that distinguish positive responders as well as the subsequent identification of targets for perturbation. Citation Format: Salil S. Bhate, Nikolay Samusik, Yury Goltsev, Garry P. Nolan. Automatic identification of cell niches and immune interactions important for clinical outcomes using multiparameter imaging and deep neural networks [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR14.

Abstract 3307: Improved survival in AML by in vivo drug targeting of conserved regulatory pathways in phenotypically distinct tumor-initiating compartments

Increasing evidence suggests tumors are maintained by cancer stem cells, however their nature remains controversial. In a HoxA9-Meis1 (H9M) driven model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy–stem/progenitor cells (Lin − kit + ), and committed progenitors of the myeloid (Gr1 + kit + ) and lymphoid lineages (Lym + kit + ). Each compartment clonally recapitulated the original range of tumor cell immunophenotypes in vivo, including cells with a less-differentiated immunophenotype. These distinct populations largely shared signaling networks, and in vivo pharmacologic targeting of shared pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, these data show that H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, in some malignancies, tumor-initiation ability (or “cancer-stemness”) can represent a targetable, cellular state that can exithat exists independently of distinct immunophenotypic definition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3307. doi:1538-7445.AM2012-3307

Abstract B6: Identification of novel mutations in the inhibitory adaptor protein LNK in patients with JAK2 V617F-negative and -positive chronic myeloproliferative neoplasms

Dysregulated JAK-STAT signaling is a hallmark of myeloproliferative neoplasms (MPNs), as evidenced by the identification of activating mutations in JAK2, and the thrombopoietin (TPO) receptor MPL in a subset of MPN patients. Clinical trials with highly specific inhibitors of JAK2 are currently ongoing, and clinical responses have been observed in the majority of MPN patients, validating JAK2 as an important therapeutic target in these patients. In addition, responses have been observed in patients lacking known mutations in JAK2 or MPL, suggesting that other regulatory elements in this pathway are altered. However, the molecular basis for this observation is not well understood. One regulator of JAK-STAT signaling is LNK (SH2B3), a member of a family of adaptor proteins that share several structural motifs, including a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain, an SH2 domain, and a conserved C-terminal tyrosine residue. LNK binds to MPL via its SH2 domain and co-localizes to the plasma membrane via its PH domain. Upon cytokine stimulation with TPO, LNK binds strongly to JAK2 and inhibits downstream STAT activation, thereby providing critical negative feedback regulation. LNK-/- mice exhibit an MPN phenotype, including an expanded hematopoietic stem cell compartment, megakaryocyte hyperplasia, splenomegaly, leukocytosis, and thrombocytosis. We sequenced LNK in a cohort of MPN patients, leading to the identification of novel mutations in 7/159 (4.4%) patients. One patient with JAK2 V617F-negative primary myelofibrosis (PMF) exhibited a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. Six additional patients were found to have point mutations affecting conserved residues in the PH domain. Interestingly, a point mutation leading to an E208Q substitution was found in one JAK2 V617F- negative patient with essential thrombocythemia (ET), as well as one JAK2 V617F-positive ET patient. Similarly, a P242S substitution was also found in both a JAK2 V617F-negative ET patient, as well as a JAK2 V617F-positive patient with post-polycythemic myelofibrosis. These latter findings suggest that even in the presence of the JAK2 V617F mutation, abrogation of LNK function may be a cooperating pathogenetic mutation. TPO-dependent BaF3-MPL cells transduced with the LNK DEL mutant exhibited augmented and sustained TPO-dependent growth and activation of JAK2-STAT3/5. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes. Primary patient samples from MPN patients bearing the LNK DEL and E208Q mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant. The STAT3/5 activation response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Our identification of mutations in LNK, the first reported in human disease, demonstrates that loss of JAK-STAT negative feedback control is a novel mechanism of MPN pathogenesis. As each of these LNK mutations localizes to the PH domain and appears to be heterozygous, mislocalized mutant LNK may exert a dominant negative effect by binding and sequestering wild-type LNK. These findings may also partly explain why some MPN patients lacking JAK2 or MPL mutations respond to treatment with JAK2 inhibitors, and highlight the importance of a more complete understanding of the role of inhibitory pathways in MPN pathogenesis. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B6.

Abstract 239: Mutation of the inhibitory adaptor protein LNK drives potentiated JAK-STAT signaling in patients with JAK2 V617F-negative myeloproliferative neoplasms

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Dysregulated JAK-STAT signaling via activating mutations in tyrosine kinases (e.g. JAK2 and MPL) is a hallmark of chronic myeloproliferative neoplasms (MPNs). Even in the absence of mutations in JAK2 or MPL, JAK-STAT activation can be demonstrated, suggesting that alterations of other regulatory elements in this pathway may contribute to MPN pathobiology. One regulator of JAK-STAT signaling is LNK (SH2B3), an adaptor protein that binds to MPL via it SH2 domain and co-localizes to the plasma membrane via its pleckstrin homology (PH) domain. Upon cytokine stimulation, LNK binds strongly to JAK2 and dampens or terminates downstream STAT activation. LNK−/- mice exhibit features consistent with an MPN phenotype, including splenomegaly, leukocytosis, and thrombocytosis. We therefore sequenced LNK in 34 JAK2 V617F-negative MPN patients, and report the identification of novel mutations in exon 2 of LNK in two patients. In a patient with primary myelofibrosis, a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains was identified. A second patient with essential thrombocythemia exhibited a missense mutation leading to an E208Q substitution in the PH domain. DNA isolated from cultured skin fibroblasts revealed wild-type (WT) sequence, confirming that these mutations were somatic. TPO-dependent BaF3-MPL cells were transduced with WT and mutant LNK. While WT LNK inhibited TPO-dependent growth and activation of JAK2-STAT3/5, the DEL mutation led to loss of these negative feedback properties, thereby permitting augmented and sustained JAK-STAT activation in response to TPO stimulation. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes. In peripheral blood samples obtained from MPN patients, stimulation with TPO or G-CSF revealed a unique phosphorylated STAT3/5 (pSTAT3+/5+) subpopulation that was increased in DEL compared with normal donor samples. A similar pSTAT3+/5+ subpopulation was seen with JAK2 V617F and MPL W515L-positive samples, suggesting that this may be a shared feature of MPNs. E208Q cells exhibited STAT3/5 phosphorylation in response to TPO, but not G-CSF, indicating that a partial loss of LNK function may generate differential STAT activation profiles in response to specific cytokines. The cytokine-responsive pSTAT3+/5+ cells from DEL were primarily CD34+, and the DEL mutation was detected in this subset, suggesting that LNK mutations arise in a hematopoietic stem or progenitor cell. Finally, the pSTAT3+/5+ response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Thus, mutations in LNK, the first reported in human disease, lead to loss of LNK negative feedback function and represent a novel mechanism of MPN pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 239.