scholarly journals Co-Targeting E-Selectin/CXCR4 with GMI-1359 Facilitates AML Stem Cell Mobilization and Protects BM Niches from Anti-Leukemia Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3348-3348
Author(s):  
Kyung Hee Chang ◽  
Tomasz Zal ◽  
Mahesh Basyal ◽  
Lauren Ostermann ◽  
Muharrem Muftuoglu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Targeted Therapy ◽  
Signaling Pathways ◽  
Research Funding ◽  
Leukemia Cells ◽  
Publicly Traded ◽  
Selectin Ligand ◽  
Dual Inhibition ◽  
Survival Signaling ◽  
Combinatorial Treatment

Abstract Acute myeloid leukemia (AML) is characterized by the heterogeneous clonal expansion of undifferentiated myeloid cells in the bone marrow (BM). AML cells compete with normal hematopoietic cells and rewire the BM microenvironment into niches that selectively support leukemia stem cells (LSC). The leukemic niche produces soluble factors that facilitate the retention of LSC and provide protection from cytotoxic and targeted agents. The vascular adhesion molecule, E-selectin is expressed on endothelial cells (EC) in the perivascular niche where therapy-resistant AML cells have an increased affinity to E-selectin compared to normal hematopoietic stem cells (HSC) (Winkler et al., 2020). We previously demonstrated (Chang et al., ASH 2020) that E-selectin blockade by the pharmacological antagonist, GMI-1271 (uproleselan; GlycoMimetics, Inc) sensitized therapy-resistant LSC to Bcl-2 targeted therapy. Efficacious eradication of LSC in the BM however requires blocking multiple receptors and/or associated signaling pathways. A more optimal dislodgement of LSC from the BM could be attained by combining an E-selectin antagonism with blockade of the CXCR4/SDF-1α axis. The dual antagonist of E-selectin and CXCR4, GMI-1359 (GlycoMimetics, Inc.), has been tested in a phase 1 clinical trial (NCT02931214). Previously, we showed that GMI-1359 in combination with a FLT3-ITD inhibitor, improved survival in a xenograft model of FLT3-ITD + AML (Zhang et al., 2016). Hence, we hypothesized that co-targeting E-selectin/CXCR4 more efficiently mobilizes AML cells from BM niches and synergizes with the anti-leukemia activity of venetoclax/hypomethylating agent (Ven/HMA). Intra-vital 2-photon imaging and tracking of individual leukemia cells in triple reporter mice (Blood: dextran-TRITC; Host T-cells: DsRed; Host myeloid CD11 cells: EYFP) injected with AML cells carrying a turquoise fluorescent protein reporter gene suggested that dual inhibition of E-selectin/CXCR4 with GMI-1359 significantly enhanced AML cell motility (Fig 1. from 2.2 um/min to 5.4 um/min, p<0.001). Individual cells were dislodged from the niche and traveled long-distance. The combined inhibition of E-selectin and CXCR4 depleted BM leukemia cells in vascular niche areas. In a patient-derived primary AML xenograft (PDX) model (harboring mutations in JAK2 and c-Kit), combinatorial treatment of GMI-1359 with Ven/HMA significantly reduced BM retention of LSC compared to control cohorts or to Ven/HMA alone (p = 0.02 and p=0.003, respectively). In order to better understand how the augmented AML mobilization improves the efficacy of AML therapy, BM cells from PDX mice treated for 2 weeks with GMI-1271, GMI-1359, Ven/HMA, and their combinations were analyzed by single-cell proteomics (CyTOF). Blockade of E-selectin alone or dual E-selectin/CXCR4 inhibition in combination with Ven/HMA diminished levels of E-selectin ligand, mTOR, pFAK, pRb, cMyc, while increasing p21 and cleaved caspase3, which was associated with significant reduction of BM-resident LSC compared to Ven/HMA alone (CD45+34+CD38-CD123+, p= 0.03). AML blasts from the BM of the combinatorial treatment groups showed altered signaling including decreased Ki67, pRb, pNFkB, pPI3K, and E-selectin ligand, and increased levels of cleaved caspase 3. We further found that Ven/HMA significantly diminished CD31+ EC in the BM compared to control cohorts (p= 0.009). However, pharmacological antagonists of E-selectin or E-selectin/CXCR4 protected EC from Ven/HMA-induced detrimental insults through upregulation of survival signaling cascades including pAKT, pERK, pMAPK and decreased eNOS expression in EC compared to Ven/HMA treatment alone. Both EC and MSC were protected by dual inhibition of E-selectin/CXCR4 with GMI-1359. We also observed upregulated pro-survival signaling pathways such as phosphorylation of AKT-MAPK-ERK along with increased Bcl-xL, Bcl-2, and Idu expression in MSC from the GMI-1359 + Ven/HMA treated PDX mice compared to Ven/HMA single treatment cohorts. Collectively, our results provide strong evidence that co-targeting E-selectin/CXCR4 with GMI-1359 profoundly reduces BM retention of LSC as well as protects BM niche component cells from apoptosis induced by targeted therapy, resulting in improving the anti-leukemia activity of Ven/HMA therapy in AML. Figure 1 Figure 1. Disclosures Fogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Carter: Ascentage: Research Funding; Syndax: Research Funding. Andreeff: Oxford Biomedica UK: Research Funding; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Aptose: Consultancy; Glycomimetics: Consultancy; Medicxi: Consultancy; Senti-Bio: Consultancy.

scholarly journals Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Weiguo Zhang ◽  
Kyung Hee Chang ◽  
Mahesh Basyal ◽  
Yannan Jia ◽  
Lauren B Ostermann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Underlying Mechanism ◽  
Leukemia Cells ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Pdx Model ◽  
Selectin Ligands

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p < 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p < 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p < 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

scholarly journals NOTCH and CAR Signaling Control T Cell Lineage Commitment from Pluripotent Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Sjoukje van der Stegen ◽  
Pieter Lindenbergh ◽  
Roseanna Petrovic ◽  
Benjamin Whitlock ◽  
Raedun Clarke ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Lineage ◽  
Lineage Commitment ◽  
Publicly Traded ◽  
Car T Cell ◽  
Car T ◽  
Single Positive

Chimeric Antigen Receptor (CAR) T cells are a new treatment paradigm for relapsed/refractory hematopoietic malignancies. However, their autologous nature imposes manufacturing constraints that can delay CAR T cell availability and increase their cost. We previously established proof of principle that αβ T cell-derived induced pluripotent stem cells (TiPSCs) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013). The use of cloned TiPSC further enhances the feasibility of verifying genome integrity of the genetically engineered stem cells and should in principle yield highly homogenous cell products. Using αβ T cell-derived TiPSCs transduced with a well-defined CD19-specific CAR (1928z; Park, NEJM, 2018), we previously demonstrated that TiPSCs can be differentiated into CAR T cells. These T cells retained their endogenous T cell receptor (TCR) and also displayed characteristics of innate lymphoid cells. We have now examined how the timing of CAR expression as well as the CAR signaling strength influence T cell lineage commitment, enabling better control towards αβ T cell lineage commitment. αβ T cell lineage development depends in part on a precisely orchestrated interactions between NOTCH and (pre)TCR signaling, the timing and strength of which are crucial for αβ lineage commitment. Because TiPSCs harbor rearranged TCRα and TCRβ genes, mature TCR expression occurs earlier than if it required VDJ recombination, skewing differentiation towards acquiring innate features including CD4-CD8- double-negative or CD8αα single-positive phenotypes. We show that providing strong NOTCH stimulation counteracts the effects of early antigen receptor expression, facilitating CD4+CD8αβ+ double positive (DP) formation. We hypothesized that CAR signaling in the absence of ligand binding (tonic signaling) may mimic a TCR signal, the strength and timing of which could re-direct lineage commitment. We therefore investigated CARs providing different levels of signaling strength and the impact of delaying the onset of CAR expression. Tonic CAR signaling was measured in peripheral blood T cells expressing 1928z or 1928z-1XX, a construct in which the second and third ITAM in the CD3ζ domain have been mutated to be non-functional (Feucht, Nat Med, 2019), following either retroviral transduction (SFG vector) orTRAC-targeted cDNA integration, placing CAR expression under the transcriptional control of the TCRα promoter (Eyquem, Nature, 2017). CAR signaling in the absence of antigen exposure, measured by phosphorylation of ITAM3, ERK1/2 and ZAP70, was reduced by bothTRAC-targeting and reduction of functional ITAMs, with additive effects when combined inTRAC-1928z-1XX. Three of these engineering strategies (virally expressed 1928z,TRAC-1928z andTRAC-1928z-1XX) were evaluated in the context of TiPSC-derived T cell differentiation. Virally expressed 1928z (resulting in constitutive CAR expression throughout differentiation) resulted in the predominant generation of innate-like CD8αα T cells, associated with the absence of early T cell lineage markers such as CD5, CD2 and CD1a. Delayed expression of 1928z throughTRACtargeting resulted in increased CD5, CD2 and CD1a, but did not yield any more CD4+CD8αβ+ DP cells. In TiPSC expressingTRAClocus-encoded 1928z-1XX, a greater DP population emerged, from which CD8αβ single-positive T cells could be induced. Phenotypic analyses of clonal TRAC-1928z-1XX TiPSC lines further establish the interplay between CAR and NOTCH1 in determining αβ lineage commitment. Together these data show that early TCR and CAR expression skew T cell lineage commitment towards an innate-like T cell fate, which can be overcome by controlling the strength and timing of NOTCH, TCR and CAR signaling. These studies pave the way for the predetermined generation of a variety of CAR T cell types endowed with different functional attributes. Disclosures Whitlock: Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Clarke:Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Valamehr:Fate Therapeutics, Inc:Current Employment, Current equity holder in publicly-traded company.Riviere:Juno Therapeutics:Other: Ownership interest, Research Funding;Takeda:Research Funding;Fate Therapeutics Inc.:Consultancy, Other: Ownership interest , Research Funding;FloDesign Sonics:Consultancy, Other: Ownership interest;Atara:Research Funding.Sadelain:Atara:Patents & Royalties, Research Funding;Fate Therapeutics:Patents & Royalties, Research Funding;Mnemo:Patents & Royalties;Takeda:Patents & Royalties, Research Funding;Minerva:Other: Biotechnologies , Patents & Royalties.

Reverse Phase Protein Analysis of Mesenchymal Stem Cells from AML Patients and Healthy Donors Reveals Distinct Patterns of Protein Expression Reflecting Differences in Senescence, Differentiation and Survival Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4376-4376 ◽  
Author(s):  
Peter P. Ruvolo ◽  
Venkata Lokesh Battula ◽  
YiHua Qui ◽  
Vivian R Ruvolo ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Leukemia Cells ◽  
Reverse Phase ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Phosphorylated Akt ◽  
Phase Protein ◽  
Survival Signaling

Abstract The bone marrow microenvironment (BME) critically supports hematopoietic stem cells and protects leukemia cells from chemotherapy, immune surveillance, and related stresses. A critical component of the BME is the mesenchymal stem cell (MSC). Dan Link’s group demonstrated that MSC are essential for human hematopoiesis, particularly as a source of SDF-1, which regulates homing, proliferation, and differentiation of HSC. Moreover, studies from our group and others have demonstrated that MSC protect leukemia cells from chemotherapy. At present, very little is known about MSC derived from AML patients, and an understanding of the proteomic makeup of these cells in the leukemia microenvironment could help to elucidate mechanisms involved in supporting their pro-tumor function. We used reverse phase protein array analysis (RPPA) to compare the expression of 151 proteins in MSC derived from AML BMs (N = 106) with those from healthy donors (N = 71). The expression of 45 of these proteins was deemed significantly different (p < 0.01) between the two sets. AML MSC expressed higher levels of p53 and p21 (CDKN1A), and the expression of the latter was correlated with other proteins within each MSC set. Using beta-galactosidase staining, AML MSC were found to undergo senescence more frequently than normal MSC. Elevated p21 in AML MSC is consistent with this finding. While 15 proteins were positively, and 20 proteins negatively, correlated with p21 expression in normal MSC, there were only three proteins positively, and nine negatively, correlated in AML-derived MSC. In normal MSC, SMAD1 (a key component in MSC growth and differentiation involving multiple receptors like TGF beta and BMP) expression and AKT signaling were low when p21 is expressed. However, in AML MSC this association was not seen, albeit a negative correlation with ITGAL was observed. SMAD1 expression was higher in normal MSC. In normal MSC, the expression of SMAD1 was negatively correlated with PPARG and NPM1, and was positively correlated with the expression of phosphorylated ELK. The opposite relationship was seen in AML MSC (i.e., PPARG and NPM1 exhibited positive correlation with SMAD1 and phosphorylated ELK was negatively correlated with the protein). While the significance of these relationships remains to be determined it is interesting to note that PPARG is a key regulator of adipocyte differentiation in MSC, so perhaps this alteration of SMAD/PPARG in AML MSC could impede their differentiation potential. In an accompanying abstract from our group, we report that AML MSC are primed toward osteoblastic differentiation and do not differentiate into adipocytes (Battula VL et al, ASH 2014). The RPPA data on PPARG is consistent with this finding. SMAD1 also positively regulates miR-21. Since p21 is a miR-21 target, it seems possible that the differences in expression could be attributed to SMAD1 and miR-21 signaling. We analyzed miR-21 expression in normal and AML-derived MSC (N = 10, each) using qRT-PCR and found a statistically significant (p =0.014) increase in its expression in normal MSC relative to their disease counterparts. When anti-miR-21 was transduced into healthy donor MSC, which caused a 3-fold increase in p21 (but no difference in cyclin D1 expression, another miR-21 target whose expression was also increased in AML MSC). AML MSC also exhibited higher protein expression of the B55 alpha subunit (PPP2R2A) of protein phosphatase 2A (PP2A). This expression contrasted interestingly with that of leukemia cells, since we have previously reported low PPP2R2A levels in AML blasts associated with shorter remission durations (Ruvolo et al Leukemia 2011). Furthermore, AKT phosphorylation was negatively correlated with PPP2R2A expression in AML blasts, and normal MSC, but there was no correlation between PPP2R2A and phosphorylated AKT in AML MSC. Also, expression of PPP2R2A was positively correlated with the expression of the survival protein NOL3 (ARC) which may provide new clues to possible survival mechanisms in AML MSC. In summary, these findings represent insights into the proteomic profiling of normal and AML MSC. Results suggest that senescence (via p21), differentiation potential (involving SMAD/PPARG pathway), and survival signaling (including PP2A/AKT) are altered in AML MSC. Studies are underway to determine how these variations in MSC properties impact the AML microenvironment. Disclosures Carter: Tetralogic Pharmaceuticals: Research Funding.

scholarly journals A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3308-3308
Author(s):  
Bianca L Rivera ◽  
Shanisha Gordon ◽  
Srinivas Aluri ◽  
Yang Shi ◽  
Samarpana Chakraborty ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transgenic Mice ◽  
Rna Sequencing ◽  
Murine Model ◽  
Research Funding ◽  
Publicly Traded ◽  
Gfp Expression ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Stop Cassette

Abstract Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders. Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs. To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies. Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. Figure 1 Figure 1. Disclosures Frank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

scholarly journals Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 778-778
Author(s):  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Twee Tsao ◽  
Yihua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Mtor Signaling ◽  
Leukemic Cells ◽  
Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

scholarly journals Matched Targeted Therapy for Pediatric Patients with Relapsed, Refractory or High-Risk Leukemias: A Report from the LEAP Consortium

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 261-261 ◽  
Author(s):  
Yana Pikman ◽  
Sarah K. Tasian ◽  
Maria Luisa Sulis ◽  
Todd M Cooper ◽  
Melinda Pauly ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
High Risk ◽  
Research Funding ◽  
Drug Testing ◽  
Genetic Alterations ◽  
Tumor Board ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Tier 1

Abstract Despite the remarkable pace of characterizing the genomics of pediatric acute leukemias, the integration of real-time sequencing results into clinical practice has lagged. With increased availability of molecularly-targeted therapies, the promise of matching genetic lesions in patients' leukemia cells to treatment has not yet been fully realized. We established the first pediatric leukemia clinical genomics consortium in the United States, known as the Leukemia Precision-based Therapy (LEAP) Consortium, which includes 13 major pediatric cancer institutions. We hypothesized that it is feasible to identify and match, in real-time, actionable alterations with a targeted therapy for pediatric patients with relapsed, refractory or high-risk leukemias or myelodysplastic syndrome (MDS). Using a combination of a DNA-based next-generation sequencing panel and RNA-based gene fusion testing, followed by data review by our multidisciplinary molecular tumor board, we are conducting a clinical trial to test this hypothesis. To date, we have enrolled and reviewed data from 143 patients stratified by disease status: Cohort 1, patients with relapsed or refractory leukemias (n=93), and Cohort 2, patients with de novo high-risk leukemias or MDS (n=50). A matched targeted therapy (MTT) recommendation has been made for 72% (n=103) of patients, tiered based on the level of evidence linking the mutation to potential activity of targeted therapy in the context of each patient's disease (Tier 1: 11%, Tier 2: 4%, Tier 3: 41%, Tier 4: 6%, Tier 5: 10%). Of the 44 patients in Cohort 1 with clinical follow-up data, 5 (11%) had alterations in therapy made based upon sequencing results and MTT recommendation. These include the use of the MEK inhibitor trametinib for RAS mutant acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (n=2), dasatinib for B-ALL and T-ALL with NUP214-ABL1 translocations (n=2) and ponatinib for B-ALL with an ABL1 T315I mutation (n=1). In addition, this genomic data refined diagnosis and supported additional germline assessment in a subset of patients. In parallel to the genomic sequencing, we are conducting high-throughput drug sensitivity (HTS) assays to study in vitro anti-leukemia effects of a panel of up to 120 targeted inhibitors in the context of leukemia-associated genetic alterations. Of the initial 106 accrued patients, 40 (38%) had adequate amounts of blood or bone marrow for in vitro drug testing. All samples were tested in the inhibitor panel assay, and 65% of the samples yielded interpretable results. Inhibitor screening data was compiled for all 12 patients with genetic alterations resulting in Tier 1, 2 or 3 MTT recommendations. This subset of patients had leukemias with two distinct molecular profiles: 1) oncogenic RAS signaling pathway mutations or 2) oncogenic tyrosine kinase alterations. Our molecular tumor board recommended trametinib for the first group of patients and tyrosine kinase inhibitors (TKIs), specific to the mutation, for the second group of patients. In vitro HTS data analysis demonstrated dose-response sensitivity of leukemia cells with RAS pathway mutations to trametinib, many of which had half-maximal inhibitory concentration (IC50) less than 50 nM. Similarly, leukemia cells from patients with FLT3, ABL1 or KIT mutations generally showed dose-responses to relevant TKIs with low IC50s. Overall, HTS data were concordant with MTT recommendations informed by the sequencing results. This first in the US multi-institutional prospective leukemia genomics trial brings state-of-the-art clinical genetic testing, assessment of prognostic biomarkers, selection of patients for germline testing, and targeted therapeutic treatment recommendations to children and young adults with high-risk, relapsed or refractory leukemias or MDS. Our collaboration provides a unique opportunity to perform sophisticated patient-specific in vitro drug testing assays to impact MTT discovery efforts, which we plan to validate in vivo using patient-derived xenograft models where feasible. We believe that a model like the LEAP Consortium has the potential to transform precision medicine approaches for children with high-risk leukemias and to inform future genomics-guided therapeutic trials and drug discovery efforts. Disclosures Tasian: Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding. Burke:JAZZ: Speakers Bureau; Shire: Speakers Bureau; AMGEN: Speakers Bureau. Tyner:AstraZeneca: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Janssen: Research Funding; Array: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Genentech: Research Funding.

scholarly journals Functional Genomics and Computational Approaches Identify Novel Small Molecules Targeting Quiescent Leukemia Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1391-1391
Author(s):  
Costakis Frangou ◽  
Jason Den Haese ◽  
Jordan Warunek ◽  
Scott Portwood ◽  
Norma J Nowak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Signaling Pathways ◽  
Research Funding ◽  
Cell Tracking ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice

Abstract Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients with leukemia; however, many will ultimately die because of disease relapse and development of drug resistance. Leukemias are cancers of the blood cells that result from alteration of the normal physiological constraints that regulate hematopoietic stem cells (HSCs). General characteristics of leukemia stem cells (LSCs) such as self-renewal, self-protection and proliferative quiescence represent inherent mechanisms that at least partially explain drug resistance and recurrence in post-therapy leukemia patients. Acute myeloid leukemia (AML) is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. Several recent studies suggest that this heterogeneity extends to LSCs and can vary between patient subgroups, and even within individual patients. Moreover, the complexity of AML is further complicated by the existence of functionally diverse leukemic and preleukemic clones. Accordingly, the hierarchical organization of AML suggests that this may be relevant to current therapies that primarily target proliferating progenitors/blast cells, which lack self-renewal capacity, and not LSCs. In the current study, we rationalized that understanding how LSCs differ from normal HSCs at the molecular level, is an essential first step towards developing novel targeted therapies and achieving permanent disease remission. Despite the identification of novel LSC-specific markers, there is considerable heterogeneity in expression of these markers amongst AML patients. However, in addition to marker-enrichment strategies, LSCs can be identified by virtue of their quiescent and slow-cycling properties. For example, label-retaining cells can be isolated and used in functional assays but significant technical limitations impede broad utility of this approach. To this end, we describe the development and use of novel multi-fluorescent protein markers and DNA bar codes integrated into the cellular genomes by lentivirus, as single-cell tracking devices for monitoring LSCs in vivo. We demonstrate how LSCs can transition between a "proliferation phase" and a "quiescence phase" in vivo. Furthermore, using high-throughput quantitative transcriptome sequencing (Q-RNA-Seq) and RNAi genetic perturbation's focusing on well-defined self-renewal signaling pathways, we develop a differential network-based model to identify LSC-specific genes and subsequently prioritize/rank candidates as potential drug targets. In the current study, we identify several molecular targets deregulated in quiescent versus proliferating LSCs and a mutual set of signaling pathways that facilitate leukemic transformation downstream of diverse initiating mutations/lesions. Remarkably, both quiescent and dividing LSCs but not HSCs, were 'addicted' to SSRP1 - an essential component of the ubiquitous FACT chromatin remodeling complex. Two orally available quinacrine-related DNA-intercalating compounds inhibiting function of FACT (CBL0100 and CBL0175, respectively) suppressed LSC proliferation in vitro and in vivo, as demonstrated by production of leukemic clonogenic cells (CFU) and long-term engraftment of immunodeficient NSG mice, by simultaneous inhibition of NF-kB (stimulated and basal forms) and activation of p53. Furthermore, in a secondary transplantation experiment, leukemic cells obtained from CBL0175 treated mice (primary) failed to engraft into secondary NSG mice in a serial transplantation model by selectively targeting the LSC compartment. Collectively, we present a novel network-based polypharmacology approach that provides unique opportunities to preferentially ablate LSCs (quiescent and dividing types), with potentially profound clinical implications. Disclosures Frangou: Cellecta: Employment. Portwood:ImmunoGen: Research Funding. Wang:ImmunoGen: Research Funding.

scholarly journals PI3-Kinase Deletion Dysregulates Autophagy in HSCs and Promotes Myelodysplasia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 323-323
Author(s):  
Kristina Ames ◽  
Imit Kaur ◽  
Shayda Hemmati ◽  
Shira Glushakow-Smith ◽  
Lindsay Meg Gurska ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Research Funding ◽  
Akt Pathway ◽  
Publicly Traded ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Autophagic Degradation

Abstract Myelodysplastic Syndrome (MDS) is a heterogeneous clonal malignancy arising in hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis, cytopenias, and the potential to progress to acute myeloid leukemia (AML). However, the perturbations in HSCs that lead to MDS initiation are poorly understood. It has been reported that HSCs are particularly dependent on autophagy for the maintenance of differentiation and self-renewal. We observed that, compared to healthy donor bone marrow hematopoietic stem and progenitor cells (HSPCs), MDS patient stem and progenitor cells (Lin-CD33-CD34+CD38-) have abnormal levels of autophagic degradation, as demonstrated by abnormal intracellular LC3II and P62 staining (Figure 1A). Autophagy is known to be regulated by the (PI3K)/AKT pathway, which transduces hematopoietic growth factor and cytokine signals in HSCs. PI3K/AKT is frequently activated in AML, but its role in MDS is less clear. Surprisingly, we found that CD34+ cells from a subset of MDS patients have upregulated expression of PTEN, the major negative regulator of the PI3K/AKT pathway, suggesting that PI3K/AKT may be downregulated in MDS stem cells. Therefore, we hypothesized that the Class IA PI3K isoforms (P110α, β, and δ) are required to maintain HSC differentiation and self-renewal. To understand the consequences of PI3K downregulation in HSCs, we generated a triple knockout (TKO) mouse model with conditional deletion of P110α and P110β in hematopoietic cells, and germline deletion of P110δ. Surprisingly, we found that PI3K deletion causes transplantable pancytopenia and decreased survival, despite the abnormal expansion of donor TKO HSCs (Figure 1 B,C). Consistent with this inefficient hematopoiesis, TKO bone marrow cells exhibited dysplastic features in multiple blood lineages and multiple chromosomal abnormalities (Figure 1 E,F), suggesting that PI3K inactivation in HSCs can promote MDS initiation. To determine whether impaired HSC differentiation in TKO mice could be due to dysregulated autophagy, we assessed autophagy in TKO HSCs by flow cytometry and immunofluorescence with the autophagosomal marker, LC3II. Our results showed that, compared to the WT controls, TKO HSCs have inefficient autophagic flux and decreased degradation of the cargo protein P62. We also discovered that TKO HSCs have significantly enlarged autophagic vesicles (Figure 1 G), and impaired fusion of autophagosomes with lysosomes, consistent with a marked defect in autophagic degradation. Treatment of TKO mice with two pharmacologic inducers of autophagy, rapamycin or metformin, improved HSC differentiation with an increase in Flk2+ MPPs (Figure 1 H), reduced dysplasia, and decreased the size of the TKO mutant clone in chimeric mice. Thus, our results uncover an important role for PI3K in regulating autophagy in HSCs to maintain the proper balance between self-renewal and differentiation. Our new mouse model of MDS will be a useful tool to study the mechanisms of MDs initiation. In addition, our findings open exciting avenues for future investigations of autophagy-inducing agents in MDS. Figure 1 Figure 1. Disclosures Verma: Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; BMS: Research Funding; GSK: Research Funding. Gritsman: iOnctura: Research Funding.

scholarly journals High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Relapsed/Refractory AML By Concomitant Analysis of Cell Surface Antigens, Signaling Pathways and Anti-Apoptotic Proteins

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-26
Author(s):  
Muharrem Muftuoglu ◽  
Zoe Alaniz ◽  
Duncan Mak ◽  
Angelique J. Lin ◽  
Jared K. Burks ◽  
...  
Keyword(s):  
Single Cell ◽  
Signaling Pathways ◽  
Research Funding ◽  
Expression Patterns ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
High Dimensional ◽  
Therapeutic Approaches ◽  
Car T ◽  
Immune Phenotypes

Background: The heterogeneous and adaptive nature of AML-associated genomic and proteomic landscape may account for disease relapse and poor prognosis, as therapy-associated selective pressure drives the emergence and expansion of AML clones with features different from those detected at diagnosis. The evolving focus is on single-cell analytical tools to fully capture the pathobiological heterogeneity of AML. We leveraged CyTOF to interrogate the proteomic heterogeneity in patients with R/R AML, which could potentially permit the design of rational combinatorial therapeutic approaches targeting vulnerabilities in these cells.. Methods: To dissect AML heterogeneity and its contribution to treatment failure, we designed a 51-parameter CyTOF panel and interrogated cellular hierarchy, major immune phenotypes, anti-apoptotic molecules, signaling pathways, exhaustion markers and attractive targets for CAR T-cell therapy in R/R AML (n:13). Results: First, we generated two-dimensional t-SNE maps and observed that the leukemic bone marrow compartment harbored immature (CD34+CD38- and CD34+CD38+) and mature leukemic blasts (CD33+CD34-) and major immune subsets. Constitutively active signaling pathways characterized by high levels of p-4EBP1, p-MEK1/2, p-S6 and p-AKT, marked immature and mature leukemia cells and comparative analysis revealed that monocytic blasts harbored more active signaling networks. The proportions of these subpopulations varied significantly across patients. We initially assessed the distribution of anti-apoptotic molecules across these leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and CD68 expressing leukemic blasts with monocytic differentiation had significantly lower levels of Bcl-2. This suggests that differentiated leukemic cells could preferentially survive under selection pressure of Bcl-2 inhibitors. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemia cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors (Venetoclax and AZD5991) resulted in highly synergistic effects in AML PDX models. Hence, this analysis supports the hypothesis that Mcl-1 overexpression is a resistance factor to Bcl-2 inhibition, usually understood as developing in the same cell. Next, we assessed expression patterns of putative CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 was uniformly expressed across all leukemia compartments, but was not ubiquitously expressed in all patient samples, revealing substantial interpatient heterogeneity in R/R AML and highlighting the concept of targeting at least two antigens concomitantly. Lastly, we sought to undertake high-dimensional assessment of immune compartments to identify major immune phenotypes in heavily treated R/R AML patients and discover the link between immune phenotypes and AML-associated traits. In line with leukemia cell heterogeneity, we found a significant degree of variation in immune cell composition among patients. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively, providing a rationale for use of combinatorial immunotherapeutic approach for the treatment of AML. Conclusion: Single-cell profiling of R/R AML using CyTOF reveals significantly heterogeneous expression patterns of molecules targeted by BH3 mimetics (Bcl-2 and Mcl-1), CAR T-cells, and other antibody-based immunotherapeutic therapies. This approach provides a rationale to develop combinatorial therapeutic approaches targeting distinct leukemia sub-populations with discrete expression patterns of established and novel putative targets. An example is the combined targeting of Bcl-2 and Mcl-1, which are differentially expressed in early and more differentiated leukemia subpopulations. Disclosures Carter: AstraZeneca: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding.

scholarly journals A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3512-3512
Author(s):  
Manit Munshi ◽  
Xia Liu ◽  
Amanda Kofides ◽  
Nickolas Tsakmaklis ◽  
Maria G Demos ◽  
...  
Keyword(s):  
B Cell ◽  
Family Member ◽  
Board Of Directors ◽  
Research Funding ◽  
Marked Reduction ◽  
Wild Type ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Bcr Signaling ◽  
Survival Signaling

Abstract Activating mutations in MYD88 (MYD88 Mut) are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of diffuse B-cell lymphoma (ABC DLBCL). MYD88 is a component of the Toll-like receptor (TLR) pathway. We and others previously showed that MYD88 Mut triggers assembly of a "Myddosome" complex that leads to downstream pro-survival signaling that includes IRAK4/IRAK1 and BTK triggered NF-κB (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013) and HCK mediated BTK/NF-κB, PI3K/AKT, and MAPK/ERK signaling (Yang et al, Blood 2016; Liu et al Blood Adv. 2020). The activation of the B-cell receptor (BCR) signaling component SYK has also been observed in MYD88 Mut WM (Argyropoulos et al, Leukemia 2016). In ABC DLBCL, chronic active BCR signaling underlies SYK activation that is triggered by the SRC family member LYN (Davis et al, Nature 2010). These observations led us to explore potential drivers of BCR/SYK activation in WM. We previously reported that MYD88 Mut triggered activation of SYK in WM and ABC DLBCL cells (Munshi et al, BCJ 2020). Herein, we investigated if HCK, a SRC family member that is transcriptionally upregulated and activated by MYD88 Mut could trigger the BCR pathway through SYK activation. Since LYN is an integral part of BCR signaling, we first examined its expression and activation state in MYD88 Mut WM and ABC DLBCL cells. While MYD88 Mut TMD8, HBL-1 and OCI-Ly3 ABC DLBCL cells showed strong expression of p-LYN, such expression was absent or low in MYD88 Mut BCWM.1 and MWCL-1 cells, as well as CD19-selected bone marrow derived primary lymphoplasmacytic cells (LPCs) from WM patients. In view of the above findings, we next interrogated a direct role for HCK in mediating SYK activation. We over-expressed wild-type HCK (HCK WT) or gatekeeper mutated HCK (HCK T333M) in MYD88 Mut BCWM.1 and MWCL-1 WM cell lines, and TMD8 ABC DLBCL cells. In all these cell lines, over-expression of HCK WT or HCK T333M triggered a robust increase in phosphorylation of SYK Y525/Y526 in comparison to vector only transduced cells. Moreover, using an inducible vector system, knockdown of HCK showed a marked reduction in phosphorylation of SYK Y525/Y526 in MYD88 Mut BCWM.1 WM and TMD8 ABC DLBCL cells. We next sought to clarify if HCK and activated SYK were present in the same signaling complex. We performed co-immunoprecipitation experiments using an HCK antibody in MYD88 Mut BCWM.1, TMD8 and wild-type MYD88 (MYD88 WT) Ramos cells. The HCK antibody effectively pulled down p-SYK in MYD88 Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. To confirm whether SYK activation was a result of HCK kinase activity, we next performed rescue experiments with the HCK inhibitors A419259 and KIN-8194 in MYD88 Mut BCWM.1 and MWCL-1 WM and TMD8 ABC DLBCL cells expressing either HCK WT or the HCK T333M protein that abrogated the activity of these inhibitors against HCK. Expression of the HCK T333M protein produced marked resistance to A419259 as well as KIN-8194 versus vector or HCK WT transduced BCWM.1 and MWCL-1 cells. By PhosFlow analysis, we observed that expression of HCK T333M but not HCK WT led to persistent activation of HCK and SYK in the presence of A419259 or KIN-8194 in BCWM.1 and MWCL-1 WM cells, and TMD8 ABC DLBCL cells. Consistent with these observations, treatment of primary MYD88 mutated WM LPCs cells with either A419259 or KIN-8194 also showed marked reduction in both p-HCK and p-SYK expression by PhosFlow analysis. Taken together, our findings show that SYK is activated by HCK in MYD88 Mut B-cell lymphomas cells; broaden the pro-survival signaling generated by aberrant HCK expression in response to MYD88 Mut; and help further establish HCK as an important therapeutic target in MYD88 Mut B-cell lymphomas. Disclosures Palomba: Juno: Patents & Royalties; Rheos: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Notch: Honoraria, Other: Stock; Kite: Consultancy; Novartis: Consultancy; BeiGene: Consultancy; Priothera: Honoraria; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; WindMIL: Honoraria; Magenta: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Gray: Syros, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member) and Soltego (board member: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi: Research Funding. Munshi: Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy. Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

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