Kenneth D. Gibbs

CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells

Acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by a subset of self-renewing leukemia stem cells (LSC). We hypothesized that increased CD47 expression on human AML LSC contributes to pathogenesis by inhibiting their phagocytosis through the interaction of CD47 with an inhibitory receptor on phagocytes. We found that CD47 was more highly expressed on AML LSC than their normal counterparts, and that increased CD47 expression predicted worse overall survival in three independent cohorts of adult AML patients. Furthermore, blocking monoclonal antibodies directed against CD47 preferentially enabled phagocytosis of AML LSC and inhibited their engraftment in vivo. Finally, treatment of human AML LSC-engrafted mice with anti-CD47 antibody depleted AML and targeted AML LSC. In summary, increased CD47 expression is an independent, poor prognostic factor that can be targeted on human AML stem cells with blocking monoclonal antibodies capable of enabling phagocytosis of LSC.

Extracting a Cellular Hierarchy from High-dimensional Cytometry Data with SPADE

The ability to analyze multiple single-cell parameters is critical for understanding cellular heterogeneity. Despite recent advances in measurement technology, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system. To objectively uncover cellular heterogeneity from single-cell measurements, we present a versatile computational approach, spanning-tree progression analysis of density-normalized events (SPADE). We applied SPADE to flow cytometry data of mouse bone marrow and to mass cytometry data of human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. We demonstrate that SPADE is robust to measurement noise and to the choice of cellular markers. SPADE facilitates the analysis of cellular heterogeneity, the identification of cell types and comparison of functional markers in response to perturbations.

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.

Single-cell phospho-specific flow cytometric analysis demonstrates biochemical and functional heterogeneity in human hematopoietic stem and progenitor compartments

The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment, and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.

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.

Abstract CN07-03: CD47 is an adverse prognostic factor and therapeutic antibody target on human AML stem cells

Human acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by rare self‐renewing leukemia stem cells (LSC), which must be eliminated in order to cure the patient. We identified increased expression of CD47 on human AML LSC compared to their normal counterparts. CD47 is a cell surface molecule that serves as the ligand for SIRP‐alpha on the surface of phagocytes, which in turn transmits a dominant inhibitory signal for phagocytosis. In this way, CD47 essentially functions as a “don9t eat me” signal. We hypothesized that increased CD47 expression contributes to pathogenesis by inhibiting phagocytosis of AML LSC. Consistent with this hypothesis, we found that increased CD47 expression predicted worse overall survival in 3 independent cohorts of adult AML patients. Furthermore, we predicted that disruption of the interaction of CD47 with SIRP‐alpha would result in phagocytosis and elimination of AML LSC. We found that blocking monoclonal antibodies directed against CD47 enabled phagocytosis of AML LSC, but not normal CD34+ human bone marrow progenitor cells, by human macrophages in vitro. Additionally, coating of human AML LSC with anti‐CD47 monoclonal antibodies inhibited their engraftment in vivo in a xenotransplantation assay. Finally, analogous to a clinical therapy, treatment of human AML‐engrafted mice with anti‐CD47 antibody eliminated AML cells in the peripheral blood and bone marrow and targeted LSC. In summary, increased CD47 expression is an independent poor prognostic factor that can be targeted on human AML stem cells with monoclonal antibodies capable of stimulating phagocytosis and elimination of LSC. Targeting of CD47 with blocking monoclonal antibodies to induce phagocytosis is a novel mechanism for antibody cancer therapy. While anti‐CD47 antibodies can be effective monotherapy for human AML, such antibodies may be equally, if not more, effective as part of a combination strategy. The combination of an anti‐CD47 antibody, able to block a strong inhibitory signal for phagocytosis, with a second antibody able to bind an LSC‐specific molecule and engage Fc receptors on phagocytes, thereby delivering a strong positive signal for phagocytosis, may result in a synergistic stimulus for phagocytosis and specific elimination of AML LSC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-03.