Flt3 Ligand Research Articles

Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum‐free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)‐QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum‐free expansion media with VEGF, Flt‐3 ligand, TPO, IL‐6, and SCF. The vascular regeneration capability of MNC‐QQ cells pre‐ or post‐QQc was evaluated with an EPC colony‐forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre‐ and post‐MNC‐QQc cells from diabetic donors were injected into a murine wound‐healing model using Balb/c nude mice. The percentage of wound closure and angio‐vasculogenesis was then assessed. This study revealed vasculogenic, anti‐inflammatory, and wound‐healing effects of MNC‐QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound‐healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient‐based vascular and regenerative therapy for patients with diabetes mellitus.

FLT3 Ligand Boosts Immune Response and May Enhance Anticancer Vaccines

In a phase II clinical trial, FLT3 ligand (FLT3L) enhanced immune cell and antibody responses.

Abstract PR012: Adoptive transfer of T cells surface-tethered with IL-12 activates a natural killer/dendritic cell axis to promote antigen spreading for enhanced anti-tumor efficacy

Abstract Despite the promise of adoptive cell therapy (ACT) in the treatment of some cancer types, success against solid tumors remains elusive due to multiple immune escape pathways. T cell therapies that affect tumor microenvironment or provide adjuvant activity in addition to tumor cell lysis hold strong potential for overcoming immune escape in solid tumors. Interleukin-12 (IL-12) is a potent stimulator of innate and adaptive immune cells that holds strong potential for cancer immunotherapy, but its clinical utility has been limited by high systemic toxicities. We have previously shown that tethering an IL-12 immunocytokine to the surface of T cells prior to adoptive cell transfer (ACT) safely improves anti-tumor efficacy by promoting T cell function specifically in the tumor. Here, we demonstrate that tethered IL-12 further delivers adjuvant activity by initiating an immune cascade involving natural killer cell-mediated activation of cross-priming dendritic cells to resulting in broader anti-tumor T cell responses. In a preclinical cell therapy model tumor-specific T cells tethered with IL-12, but not the T cells alone, triggered proliferation of tumor-specific endogenous tumor infiltrating lymphocytes (TIL). This was accompanied by increased activation and infiltration of cross-presenting dendritic cells (cDC1) in tumor-draining lymph nodes (tdLN). Analyses of cDC1 subsets revealed upregulation of CD86 expression on both migratory and resident cDC1s within the tdLN, two cell types that are critical for both tumor antigen transport to tdLN and activation of naïve CD8 T cells, respectively. This activity was specific to cDC1s, as increased activation or accumulation of type 2 dendritic cells (cDC2) was not observed. Mechanistically, the IL-12 tethered T cells upregulated expression of FLT3 ligand on NK cells, a key cytokine for cDC1 recruitment and expansion. NK cell depletion ablated cDC1 tdLN infiltration and endogenous tumor infiltrating T cell expansion, indicating that recruitment and activation of endogenous tumor-specific T cells is at least partially driven by tethered IL-12-mediated activation of NK cells to recruit and expand the cross-priming dendritic cells. Together, this demonstrates that our surface tethered cytokine technology provides powerful adjuvant activity to adoptively transferred T cells and promotes activation of endogenous anti-tumor adaptive immune response, thus promoting a broad antigenic repertoire. This approach thus holds potential to help overcome challenges of antigen escape and tumor heterogeneity that limit efficacy of T cell therapies against solid tumors. This abstract is also being presented as PO078. Citation Format: Kate L. Stokes, Ditte E. Jaehger, Alvin Pratama, Holmfridur R. Halldorsdottir, Gulzar Ahmad, Jon D. Nardozzi, Katharine L. Sackton, Thomas L. Andresen, Douglas S. Jones. Adoptive transfer of T cells surface-tethered with IL-12 activates a natural killer/dendritic cell axis to promote antigen spreading for enhanced anti-tumor efficacy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR012.

FLT3-ITD Mutation and FLT3 Ligand Plasma Level Were Not Associated with One-Year Survival of Indonesian Acute Myeloid Leukemia Patients.

ObjectiveTo analyze the association of FLT3-ITD mutation and FLT3 ligand plasma level with one-year survival of Indonesian acute myeloid leukemia (AML) patients.MethodsA prospective cohort study was conducted to determine the association between FLT-3-ITD mutation and FLT3 ligand plasma level with one-year survival of Indonesian AML patients. In the study, a total of 51 AML patients were obtained from two tertiary hospitals in Indonesia from year 2018 to 2020. Inclusion criteria were de novo AML male and female patients aged ≥18 years old. Exclusion criteria were prior myelodysplastic syndrome and patients that refused to participate in the study. FLT3-ITD genotype of patients was then analyzed using PCR method while FLT3 ligand plasma level was measured using ELISA method. Patients were then followed-up for 1 year or until death occurred with survival as the measured outcome. Association between independent and dependent variable were analyzed by cox regression proportional hazard.ResultsEleven patients (21.5%) in this study had FLT3-ITD mutation. The median age of AML patients was 45 (18–71) years, and the median blast percentage was 50% (5–87%). After one-year follow-up, 33 (64.7%) patients had died. The median survival of AML patients was 6 months. Univariate analysis showed no association between FLT3-ITD mutation status (HR: 1.051 ; 95% CI: 0.483–2.286; P: 0.901) and FLT3 ligand plasma level (HR: 0.798; 95% CI: 0.347–1.837; p= 0.596), and age (HR: 1.283; 95% CI: 0.575–2.862; p= 0.542) with one-year survival of AML patients, but multivariate analysis showed association between GFR with one-year survival of AML patients in this cohort (HR: 4.053; 95% CI: 1.469–11.183; p= 0.007).ConclusionOne-year survival of AML patients in Indonesia is not affected by FLT3-ITD mutation and FLT3 ligand plasma level. However, GFR showed association with one-year survival of AML patient in this cohort study.

FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy

AbstractPoint mutations of D835/I836 of the FLT3 gene have been reported in adult acute myeloid leukemia (AML), but not in pediatric AML or acute lymphoblastic leukemia (ALL). FLT3-D835/I836 mutations were found in 6 (5.4%) of 112 children with ALL older than 1 year and in 8 (16.0%) of 50 infants with ALL. Missense mutations were found in 11 patients, 3-base pair deletions in 2 patients, and a deletion/insertion in 1 patient. Remarkably, FLT3-D835/I836 mutations were found in 8 (18.2%) of 44 infants with ALL with MLL rearrangements and in 4 (21.5%) of 19 patients with hyperdiploid ALL, but they were not found in any patients older than 1 year who had TEL-AML1 (n = 11), E2APBX1 (n = 4), or BCR-ABL (n = 6) fusion genes. Although infant ALL patients with mutations had poorer prognoses than did those without mutations, pediatric ALL patients with mutations who were older than 1 year had good prognoses. We also found FLT3-D835 mutations in 2 of 11 leukemic cell lines with MLL rearrangements. FLT3 was highly phosphorylated in these cell lines with FLT3-D835 mutations, leading to constitutive activation of downstream targets such as signal transducer and activator of transcription 5 (STAT5) without FLT3 ligand stimulation. These results suggested that FLT3-D835/I836 mutations are one of the second genetic events in infant ALL with MLL rearrangements or pediatric ALL with hyperdiploidy. (Blood. 2004;103:1085-1088)

Authentication of Primary Murine Cell Lines by a Microfluidics-Based Lab-On-Chip System.

The reliable authentication of cell lines is a prerequisite for the reproducibility and replicability of experiments. A common method of cell line authentication is the fragment length analysis (FLA) of short-tandem repeats (STR) by capillary electrophoresis. However, this technique is not always accessible and is often costly. Using a microfluidic electrophoresis system, we analyzed the quality and integrity of different murine cell lines by STR profiling. As a proof of concept, we isolated and immortalized hematopoietic progenitor cells (HPC) of various genotypes through retroviral transduction of the fusion of the estrogen receptor hormone-binding domain with the coding sequence of HoxB8. Cell lines were maintained in the HPC state with Flt3 ligand (FL) and estrogen treatment and could be characterized upon differentiation. In a validation cohort, we applied this technique on primary mutant Kras-driven pancreatic cancer cell lines, which again allowed for clear discrimination. In summary, our study provides evidence that FLA of STR-amplicons by microfluidic electrophoresis allows for stringent quality control and the tracking of cross-contaminations in both genetically stable HPC lines and cancer cell lines, making it a simple and cost-efficient alternative to traditional capillary electrophoresis.

Features of cytokine profile in patients with sarcoidosis

Sarcoidosis is an inflammatory disease of unknown etiology with damage to the lungs and other organs characterized by development of necrosis-free epithelioid cell granulomas. Granulomatous inflammation characterized by the activation of different immune systems cells, in particular T lymphocytes, and the cytokines production. Our study was aimed at investigating the characteristics of the cytokine profile of blood plasma in patients with sarcoidosis. We studied peripheral blood plasma samples of patients with sarcoidosis (n = 52). The control blood samples were taken from healthy volunteers (n = 22). The level of 46 cytokines (pg/ml) was determined, as follows: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IFNα2, IFNγ, TNFα, TNFβ, IL- 1ra, IL-10, EGF, FGF-2, Flt3 Ligand, G-CSF, GM-CSF, PDGF-AA, PDGF-AB / BB, TGFα, VEGF-A, sCD40L, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL17, CCL20, CCL22, CXCL1, CXCL8, CXCL9, CXCL10, CXCL11, CXCL13, CX3CL1. Significantly higher levels of interleukins and some proinflammatory cytokines were found in the patients with sarcoidosis, i.e., IL-3, 0.70 vs 0.20, p = 0.003; IL-4, 14.37 vs 3.15, p = 0.009; IL-5, 1.06 vs 0.89, p < 0.001; IL-12 (p70), 1.27 vs 0.56, p = 0.028; IL-17A, 1.48 vs 0.43, p < 0.001; IFNα2, 41.79 vs 25.04, p = 0.003; IFNγ, 4.13 vs 1.14, p < 0.001; TNFα, 21.67 vs 6.70, p < 0.001; anti-inflammatory cytokine IL-10, 1.03 vs 0.45, p = 0.019; growth factors: FGF-2, 40.08 vs 30.58, p = 0.008, G-CSF, 24.18 vs 8.21, p = 0.006, and VEGF-A, 42.52 vs 26.76, p = 0.048; chemokines: CCL3, 3.86 vs 1.33, p < 0,001; CCL17, 78.24 vs 26.24, p < 0.001; CCL20, 7.19 vs 5.64, p = 0.021; CCL22, 660.60 vs 405.00, p < 0,001; CXCL9, 4013 vs 1142, p < 0,001; CXCL10, 565.90 vs 196.60, p < 0.001; CXCL11, 230.20 vs 121.10, p = 0.018; CX3CL1, 56.99 vs 5.16, p < 0.001. Peripheral blood chemokine CCL11 levels were significantly lower in patients compared to the group of healthy volunteers: 77.58 vs 124.70, p = 0.022. The features of the cytokine profile in patients with sarcoidosis may indicate their important role in the processes of formation and outcomes of granulomas. These issues require an additional detailed study, comparison with phenotypes, differential course and outcomes of the disease.

Tumor Associated Macrophages Express High-Levels of FLT3 Ligand, Which Induces Activation of FLT3 Signaling That Promotes Survival of Neoplastic Cells in B-Cell Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of B lymphocytes blocked at an early stage of differentiation, which accounts for approximately 30% of childhood cancers. Almost all B-ALL blasts aberrantly express FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase involved in the development of dendritic cells and B lymphocytes. In B-ALL, the high expression of FLT3 in blasts significantly increases the odds of relapse/death in patients, indicating a pathological role of FLT3 in B-ALL. Note that gain-of-function mutations of FLT3 are relatively rare (<5%) in patients with B-ALL, suggesting an essential function of the ligand activating FLT3 in the initiation and progression of the disease. However, the presence, origin and function of FLT3 ligand (FLT3L) in B-ALL are not fully uncovered. In the present study, we observed high levels of FLT3L (234.7 ± 47.6 pg/ml) in plasma from 28 patients with B-ALL, compared with age-matched healthy donors (41.2 ± 15.5 pg/ml) by using ELISA. Among these samples, 12 were from patients achieved remission with negative minimal residue disease (MRD) but maintained high levels FLT3L (286.9 ± 71.2 pg/ml), suggesting cell-source(s) of FLT3L other than neoplastic B-ALL blasts. We hypothesized that tumor-associated macrophages (TAMs) from the microenvironment of B-ALL express FLT3L, because studies show that activated macrophages from patients with autoimmune disease express high levels of FLT3L to promote inflammation and destruction of local articular components. Previously, we cultured nurse-like cells, which are TAMs with a M2 phenotype from peripheral blood mononuclear cells (PBMCs) of patients with chronic lymphocytic leukemia (CLL) (Chen Y, et al. Blood. 2019; 134:1084-1094.). These cells are key components of microenvironment of CLL cells to support survival and migration of neoplastic B cells. In the present study, we cultured PBMCs from B-ALL patients and obtained non-neoplastic TAMs with a similar morphology to nurse-like cells. These cells were stained positive for CD68 and CD163, which are markers for M2 macrophages by immune fluorescent microscopy. We isolated RNA from purified B-ALL blasts or TAMs of the same patient for real-time PCR to measure the levels of FLT3L transcripts. FLT3L transcripts were abundant in the RNA isolated from TAMs, but were negligible in RNA isolated from B-ALL cells in each patient tested (N = 5). We also evaluated for secreted FLT3L protein via ELISA in the culture-medium of isolated TAMs and B-ALL cells with an equal cell number from 18 different patients. The cultured medium of TAMs had significantly higher concentration of FLT3L than B-ALL cells. Furthermore, we performed co-culture studies to assess the involvement of FL/FLT3 signaling in the survival of B-ALL cells cultured with TAMs. B-ALL cells co-cultured with TAMs had significantly more live cells detected by flow cytometry with annexin V and PI staining, compared with B-ALL cells cultured alone. Immunoblot showed that B-ALL cells co-cultured with TAMs had high levels of phosphorylated FLT3 and ERK, indicating the activated FLT3 signaling cascade in these cells. We speculate that the elevated level of FLT3L noted in the plasma of patients with B-ALL is produced primarily by TAMs, which reside in the microenvironment of neoplastic cells. Such high-level FLT3L may provide chemoresistance of B-ALL cells within the microenvironment. Treatment with FLT3 tyrosine kinase inhibitors may mitigate such effects, potentially enhancing the clearance of leukemia cells when used in combination with chemotherapy. Disclosures No relevant conflicts of interest to declare.

FLT3-CD3 Bispecific Antibody Specifically Eliminates Normal Hematopoietic Progenitors and AML in Humanized Mouse Models

Acute myeloid leukemia (AML) is a blood cancer arising from clonal proliferation of myeloid progenitors harboring oncogenic mutations. AML patients have poor clinical prognosis and limited therapeutic options, with myeloablation followed by hematopoietic stem cell transplantation (HSCT) as the only curative treatment. The conditioning regimens used indiscriminately kill all highly proliferative cell types, leading to life threatening side effects, and are also potentially ineffective against quiescent AML subpopulations. To address the challenges of efficacy and resistance in targeting AML, we developed a humanized bispecific antibody (CDX) that recruits T cells through CD3 to target and kill Fms-like Tyrosine Kinase 3 (FLT3) expressing cells. Normal FLT3 expression is mostly restricted to hematopoietic stem and progenitor cells (HSPCs) and its activation, through binding of FLT3 ligand (FLT3L), promotes normal differentiation of downstream blood lineages. FLT3 is also expressed on AML blasts in a majority of patients and is thought to promote survival and proliferation of AML. Although multiple promising tyrosine kinase inhibitors (TKIs) have been and are being developed to specifically target FLT3, secondary mutations leading to resistance against FLT3-TKIs remain a major obstacle. Surface expression of FLT3 in leukemic cells, as well as in HSCs, makes it an excellent target for T cell mediated conditioning that specifically eliminates both AML blasts and HSPCs, allowing for subsequent HSCT. In addition, HDX mass spectrometry revealed that our CDX binds an extracellular, membrane proximal FLT3 domain, which does not compete with FLT3L and is outside the regions commonly mutated in AML, therefore, unlike other therapies, targets all FLT3-expressing cells, regardless of mutations in the receptor. We found that in vitro treatment with CDX redirected primary human T cells to kill multiple AML-derived FLT3+ cell lines, some harboring common FLT3 mutations, at EC50 ranging from 0.3 pM to 15 pM. Additionally, the presence of FLT3L (10 nM) had no effect on the EC50 of CDX binding to FLT3+ target cells. To test CDX in vivo, we generated xenograft mice engrafted with primary human peripheral blood mononuclear cells (PBMCs) and MV4;11 cells (FLT3+ AML cell line), which were engineered to stably express EGFP. Mice were treated intravenously with 3 doses of CDX at a concentration of 0.1 mg/kg or left untreated over 1 week (n=5). Progression of AML was tracked by monitoring the frequency of EGFP-MV4;11 cells in peripheral blood (PB) of xenografted mice, which were euthanized if they presented with cachexia, hind-leg paralysis, and severe weight loss (symptoms of AML progression). Treatment with CDX significantly delayed the proliferation of MV4;11 cells, based on lower PB frequencies of EGFP-MV4;11, and increased median survival by 28 days relative to untreated mice. To more stringently model CDX efficacy in a clinical setting, we injected EGFP-MV4;11 cells into humanized mice that were generated by transplantation of human cord blood derived, CD34+ HSPCs and displayed stable multi-lineage engraftment (~20% human CD45+) with significant populations of T cells at 35 weeks. We observed that T cells in CDX-treated mice over-expressed the immune checkpoint receptor PD1, relative to untreated mice, and hypothesized that PD1 inhibition could enhance MV4;11 clearance via CDX treatment by preventing T cell exhaustion. Treatment regimens consisted of 3 doses of CDX (0.1 mg/kg), anti-PD1 antibody (100 μg), or a combination of CDX and anti-PD1 (n=5), followed by transplant with autologous mouse bone marrow cells. Treatment with CDX alone led to decreased PB frequencies of EGFP-MV4;11 cells, but only modestly improved median survival relative to anti-PD1 alone (9 days). In contrast, co-treatment with CDX and anti-PD1 antibody significantly increased median survival (16 days) and was associated with decreased PD1 expression on T cells. In addition, administration of CDX alone or in combination with PD1 in humanized mice resulted in efficient elimination of the human hematopoietic compartment from the mouse bone marrow. Based on our findings, CDX shows promise for treatment of AML with concurrent conditioning for HSCT with improved specificity compared to standard of care and even displays synergy with checkpoint inhibition of PD1. Disclosures Sirochinsky: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Shrestha:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

Preclinical Development of Anti-FLT3 CAR-T Therapy for the Treatment of Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 (FLT3) is a class III transmembrane receptor tyrosine kinase involved in survival, proliferation, and differentiation of hematopoietic stem/progenitor cells. It is preferentially expressed on the leukemic cells of myeloid lineage including acute myeloid leukemia (AML) and is mutated in approximately one-third of patients with AML, resulting in constitutive signaling associated with poor disease prognosis. Although small molecule inhibitors targeting FLT3 have shown some success in clinical trials, they only work transiently while resistance develops in virtually all patients. The only proven curative treatment for the relapsed or refractory (R/R) AML is allogenic hematopoietic stem cell transplantation (HSCT) which requires highly toxic conditioning regimens often associated with fatal side effects. Thus, there still remains an urgent need for the development of safe yet effective new therapies for the treatment of AML. We developed a novel chimeric antigen receptor modified T (CAR-T) cell therapy targeting FLT3 to eliminate FLT3+ R/R AML leukemia via cytotoxic T lymphocytes (CTL)-mediated cytolysis. Since FLT3 is also expressed on hematopoietic stem cells (HSCs) as well as on early hematopoietic progenitors (HPs), we evaluated the conditioning efficacy of our anti-FLT3 CAR-T in addition to its anti-leukemic activity. We first discovered a novel mouse monoclonal antibody that binds to the extracellular domain of human FLT3 with high affinity (0.8 nM EC50 in FLT3+ leukemic cell line REH) while not competing with FLT3 ligand in order to achieve unobstructed and efficient binding to FLT3. We next generated humanized single-chain variable fragment (scFv) antibodies and characterized their binding affinities. The scFv clone that exhibited highest binding to FLT3 (3.42 nM EC50 in REH cells) was used to design a third generation CAR construct with CD28 and 4-1BB costimulatory and CD3ζ activation domains. T cells isolated from peripheral blood (PB) were transduced with a lentiviral vector encoding the FLT3-CAR. Transduced cells exhibited stable expression of CAR protein and expanded over 120-fold after 18 days in culture. We demonstrated high cytotoxicity of FLT3-CAR-T cells towards AML-derived cell lines in co-culture experiments, even at effector-to-target cells ratios as low as 1:10. In vivo functionality of FLT3-CART was determined by flow cytometry analysis of leukemia burden in the peripheral blood of mice engrafted with GFP+ MOLM-13 (FLT3+ AML cell line) and treated with two doses of 4x106 control or FLT3-CAR-T cells. Compared to control, the appearance of MOLM-13 cells in peripheral blood was significantly delayed in FLT3-CAR-T treated mice. AML progression in mice was also assessed by detection of physical symptoms such as cachexia and hind-leg paralysis in terminal stages. FLT3-CAR-T treatment extended the median survival to 47 days compared to 24 days in control. Moreover, to test if our CAR-T therapy can also efficiently eliminate FLT3+ HSCs and HPs, humanized mice generated by engrafting human cord blood CD34+ cells were injected with autologous control or FLT3-CAR-T cells. Analysis of bone marrow 18 days post treatment, showed that mice that received FLT3-CAR-T cells exhibited dramatically lower frequencies (by 57% in CD38+ and 86% in CD38-) of human CD34+ hematopoietic stem and progenitor cells than control mice, suggesting the potential of CAR-T therapy for HSCT conditioning. In conclusion, our CAR-T therapy shows robust cytolytic activity against FLT3+ cells, demonstrates high efficacy in eradicating FLT3+ R/R AML leukemia in vivo and enables bone marrow conditioning for potentially curative HSCTs by specifically targeting FLT3+ HSCs and early HPs. To prevent the potentially harmful side effects associated with CAR-T therapies, such as cytokine release syndrome and cytotoxicity towards newly transplanted HSCs post conditioning, we are currently testing FLT3-CAR-T cells equipped with inducible caspase9 or EGFRT expression based safety switch to specifically eliminate CAR-Ts by administering FDA-approved small molecules or biologics. Disclosures Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Sirochinsky:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

FLT3 Ligand-DM1 Conjugate Selectively Targets Acute Myeloid Leukemia Cells with FLT3 Expression

Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasm featured by impaired differentiation and uncontrolled proliferation of myeloid progenitors. Gain-of-function mutations of FMS-like tyrosine kinase 3 (FLT3) present in 30-40% of patients with AML. In addition, more than 90% of AML blasts aberrantly express FLT3, making FLT3 an attractive therapeutic target for AML. Currently, several small molecule tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved in the treatment of AML, but they need to be used in combination with chemotherapy because of their limited potency to eliminate leukemic cells as single agents, largely due to the development of secondary inhibitor-resistant FLT3 mutations. Therefore, novel therapeutic strategies targeting FLT3 are needed. In the present study, we developed a FLT3 ligand-emtansine drug conjugate (FL-DM1) that targeted FLT3-positive AML cells with high potency and selectivity. We expressed recombinant human FLT3 ligand (rhFL) in the periplasm of recombinant E. Coli. The protein was purified by a two-step purification system containing Ni-NTA and Phenyl Sepharose. Our purified rhFL was bioactive to stimulate phosphorylation of FLT3 and proliferation of THP-1 cells. Next, we conjugated purified rhFL and emtansine (DM1) with SPDP linker. Reducing and non-reducing SDS-PAGE revealed that rhFL and DM1 were successfully conjugated, evidenced by a band with higher molecular weight of the conjugation product. Previous studies show that DM1 is a drug preferentially targeting proliferating cells by depolymerizing microtubules through binding at the vinca binding site of tubulin. We found that FL-DM1 reserved the physiological function of FLT3 ligand to stimulate proliferation of AML cells by inducing phosphorylation of FLT3 and the downstream signaling protein AKT in immunoblot, potentially enhancing the potency of FL-DM1 to inhibit FLT3-positive AML cells. Furthermore, flow cytometry showed that the surface expression of FLT3 significantly decreased on cells treated by FL-DM1 within two hours, which indicated the internalization of FL-DM1/FLT3 complex on these FLT3-positive AML cells, providing a mechanism of FL-DM1 entering target cells. In cell viability assay, we found that FL-DM1 effectively inhibited FLT3-positive AML cells THP-1 and MV-4-11 with IC50 around 10-30 nM. Also, FL-DM1 induced apoptosis and cell cycle arrest at G2/M phase in these cells detected by flow cytometry. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are FLT3-negative erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. We found that FL-DM1 inhibited HCD-57 cells transformed by FLT3-ITD, but not parental HCD-57 cells without FLT3 expression, indicating the selectivity of FL-DM1 to target FLT3-positive AML cells. In conclusion, our data demonstrated that FL-based drug conjugate can serve as an effective drug to target FLT3-expressing AML cells. Further studies will focus on in-vivo evaluation of FL-DM1 in animal models, the production of uncleavable SMCC linked FL-DM1 with improved in-vivo pharmacokinetic properties, and screening of FL muteins-DM1 conjugates with desired pharmacological properties. Disclosures No relevant conflicts of interest to declare.

Evolution of Gilteritinib Resistance from Residual Disease to Relapse

Evolution of Gilteritinib Resistance from Residual Disease to Relapse

Biomarker Driven Umbrella Trial of Crenolanib in Combination with Ivosidenib, Enasidenib, Venetoclax, Vyxeos and/or Salvage Chemotherapy in FLT3 Mutant AML

Biomarker Driven Umbrella Trial of Crenolanib in Combination with Ivosidenib, Enasidenib, Venetoclax, Vyxeos and/or Salvage Chemotherapy in FLT3 Mutant AML

The Impact of Sorafenib on Phospho-FLT3 Inhibition and FLT3-ITD MRD after Chemotherapy: Correlative Studies from the Phase 2 Randomized Study of Sorafenib Versus Placebo in Combination with Intensive Chemotherapy in Previously Untreated Patients with FLT3-ITD Acute Myeloid Leukemia (ALLG AMLM16)

The Impact of Sorafenib on Phospho-FLT3 Inhibition and FLT3-ITD MRD after Chemotherapy: Correlative Studies from the Phase 2 Randomized Study of Sorafenib Versus Placebo in Combination with Intensive Chemotherapy in Previously Untreated Patients with FLT3-ITD Acute Myeloid Leukemia (ALLG AMLM16)

FLT3 Inhibitors Induce Instability of p53 By Mir-181 Mediated Downregulation of Deubiquitinase YOD1 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy featured by impaired differentiation and uncontrolled proliferation of myeloid blasts. FLT3 internal tandem duplication (ITD) presents in 30-40% patients with AML, which serves as an independent poor prognostic marker and an attractive therapeutic target. Up to date, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved by FDA in the treatment of AML. However, FLT3 TKIs as single agents have limited effects to eliminate AML cells due to multiple drug-resistant mechanisms, including secondary FLT3 mutations, alternatively activated cell survival pathways, upregulation of FLT3 ligand, and downregulation of tumor suppressor genes. In the present study, we found that FLT3 TKIs decreased tumor suppressor p53 protein level by downregulation of YOD1 through miR-181 in FLT3-ITD mutant AML cells. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. By using transcriptome analysis with RNAseq, we identified multiple differentially expressed miRNAs in HCD-57 transformed by FLT3-ITD, compared with parental HCD-57 cells. miR-181a-5p and miR-181b-5p were among these highly differentially expressed miRNAs. These two miRNAs were predicted to bind in 3'UTR of deubiquitinase YOD1 by using TargetScanHuman 7.2, an online tool to predict biological targets of miRNAs. Previous studies have shown that the ubiquitination and protein level of p53 is dysregulated due to overexpressed E3 ligase MDM2/4 in AML cells, but few studies focused on deubiquitinase of p53. We found that deubiquitinase YOD1 interacted with p53 by immunoprecipitation. Overexpression of YOD1 prevented degradation of p53 led by cycloheximide, a protein synthesis inhibitor. We further found that overexpression of YOD1 resulted in decreased ubiquitination of p53, indicating that YOD1 stabilized p53 protein through deubiquitination. Subsequently, we detected the expression of miR-181, YOD1 and p53 in a FLT3-ITD positive AML cell line MV-4-11 treated by FLT3 TKIs sorafenib, sunitinib and quizartinib. Real-time quantitative PCR showed that the treatment of FLT3 TKIs upregulated the expression of miR-181a-5p/miR-181b-5p, and downregulated mRNA level of their predicted target YOD1. The mRNA level of p53 remained unchanged but its protein level decreased with enhanced ubiquitination in MV-4-11 cells treated by FLT3 TKIs in the ubiquitination assay. These data suggested that FLT3 TKIs could reduce the stability of p53 by regulating miRNA-targeted YOD1. In addition, we collected peripheral blood mononuclear cells (PBMCs) from patients with AML and age-matched healthy donors. We found increased expression of miR-181a-5p and miR-181b-5p and decreased expression of YOD1 in PBMCs from AML patients compared with healthy donors, suggesting a pathological role of miR-181 to regulate YOD1/53 pathway in AML. In conclusion, our data showed that FLT3 TKIs induced instability of p53 by miR-181 mediated downregulation of YOD1. YOD1 as a novel deubiquitinase of p53 could play important roles in drug-resistance and progression of AML. Disclosures No relevant conflicts of interest to declare.

Melanoma Evolves Complete Immunotherapy Resistance through the Acquisition of a Hypermetabolic Phenotype.

Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a "hypermetabolic" phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.

FLT3 Ligand Is Dispensable for the Final Stage of Type 1 Conventional Dendritic Cell Differentiation.

Conventional dendritic cells (cDCs) are comprised of two major subsets, type 1 cDC (cDC1) and type 2 cDC (cDC2). As each cDC subset differentially influences the nature of immune responses, we sought factors that would allow the manipulation of their relative abundance. Notably, cDC1 are less abundant than cDC2 in both lymphoid and nonlymphoid organs. We demonstrate that this bias is already apparent in bone marrow precommitted precursors. However, comparison of five common inbred strains revealed a disparity in precursor-product relationship, in which mice with fewer precursors to cDC1 had more cDC1. This disparity associated with contrasting variations in CD135 (FLT3) expression on cDC subsets. Hence, we characterized the response to FLT3 ligand during cDC1 and cDC2 lineage differentiation and find that although FLT3 ligand is required throughout cDC2 differentiation, it is surprisingly dispensable during late-stage cDC1 differentiation. Overall, we find that tight regulation of FLT3 ligand levels throughout cDC differentiation dictates the cDC1 to cDC2 ratio in lymphoid organs.

Dendritic Cells and Microglia Have Non-redundant Functions in the Inflamed Brain with Protective Effects of Type 1 cDCs

SummaryBrain CD11c+ cells share features with microglia and dendritic cells (DCs). Sterile inflammation increases brain CD11c+ cells, but their phenotype, origin, and functions remain largely unknown. We report that, after cerebral ischemia, microglia attract DCs to the inflamed brain, and astroglia produce Flt3 ligand, supporting development and expansion of CD11c+ cells. CD11c+ cells in the inflamed brain are a complex population derived from proliferating microglia and infiltrating DCs, including a major subset of OX40L+ conventional cDC2, and also cDC1, plasmacytoid, and monocyte-derived DCs. Despite sharing certain morphological features and markers, CD11c+ microglia and DCs display differential expression of pattern recognition receptors and chemokine receptors. DCs excel CD11c− and CD11c+ microglia in the capacity to present antigen through MHCI and MHCII. Of note, cDC1s protect from brain injury after ischemia. We thus reveal aspects of the dynamics and functions of brain DCs in the regulation of inflammation and immunity.

The Blood Cytokine Profile of Young People with Early Ischemic Heart Disease Comorbid with Abdominal Obesity.

Objective: The aim was to study the blood cytokine/chemokine profile of 25–44-year-old people with early ischemic heart disease (IHD) comorbid with abdominal obesity (AO). Methods: A cross-sectional medical examination of subjects in Novosibirsk, Russia, was conducted after random sampling of the above age group. A total of 1457 subjects, 804 females and 653 males, were analyzed. The epidemiological diagnosis of IHD was made in accordance with 17 validated and functional criteria, employing exercise ECG for confirmation. Simultaneous quantitative analyses of 41 cytokines/chemokines in blood serum were performed by a multiplex assay using the HCYTMAG-60K-PX41 panel (MILLIPLEX MAP) on a Luminex 20 MAGPIX flow cytometer, with additional ELISA testing. Results: Flt3 ligand, GM-CSF, and MCP-1 were significantly associated with the relative risk of early IHD. In the presence of AO, GM-CSF, MCP-1 and IL-4 also significantly correlated with the relative risk of early IHD. By univariate regression analysis, the relative risk of early IHD was associated with lowered blood concentrations of Flt3 ligand, whereas the relative risk of early IHD in the presence of AO was associated with lowered blood concentrations of GM-CSF. Employing multivariable regression analysis, only lower blood levels of Flt3 ligand were associated with a relative risk of early IHD, whereas the relative risk of early IHD in the presence of AO was limited to lower levels of IL-4. Conclusion: Findings related to Flt3 ligand, GM-CSF, and IL-4 are consistent with the international literature. Results from the present study are partly confirmative and partly hypothesis generating.

Abstract LB-292: A randomized controlled trial of yoga in men with prostate cancer undergoing radical prostatectomy

Abstract Introduction and Objectives: New diagnosis of prostate cancer (PCa) may lead to anxiety, mental distress, fear and depression in up to 30% of men, which can contribute to poor quality of life (QOL). Recent research on Yoga in cancer patients has shown significant improvement in health related QOL, emotional health and fatigue. Given the lack of randomized clinical data regarding the effects of Yoga in PCa, we performed this study to evaluate the potential impact of Yoga on disease-specific QOL in men undergoing radical prostatectomy (RP) for PCa. Material and Method: We randomly assigned newly diagnosed men with localized prostate cancer to Yoga (n = 15) or standard of care (n= 15) prior to their RP. Yoga was given to the intervention group twice a week for 6 weeks for 60 minutes before the surgery and then re-initiated 3 to 6 weeks after the surgery for another six weeks. Self-report measures of QOL [ Expanded Prostate Index Composite (EPIC), Functional Assessment of Cancer Therapy-Prostate (FACT-P), Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), Functional Assessment of Cancer Therapy-General (FACT-G) and Five Facets of Mindfulness Questionnaire (FFMQ) primary outcomes], blood specimen (cytokine and immune markers; secondary outcome) were collected at baseline, prior to the surgery and 6 weeks after surgery. Results: Improvements in EPIC sexual domain (mean difference: 8.5 points), FACT-P (mean difference: 5.3 points), FACIT-F (mean difference: 5.4 points), and FACT-G (mean difference: 7.2 points) were seen at 6 weeks in the Yoga arm compared with controls. Greatest benefit of Yoga was seen in the subdomains of EPIC sexual function (mean difference: 8.9 points), EPIC sexual bother (mean difference: 7.6 points), FACT emotional wellbeing (mean difference: 5.4 points), FACT functional wellbeing (mean difference 8.3 points) and FACT social wellbeing (mean difference 14.1 points). We identified statistically significant reduction in the inflammatory cytokines- granulocyte colony-stimulating factor (G-CSF; 0.55 [0.05 - 1.05]; p-value = 0.03), Monocyte chemoattractant protein (MCP-1; 0.22 [0.01 - 0.43]; p-value = 0.04), FMS-like tyrosine kinase-3 ligand (FLT-3 L; 0.91 [-0.01 - 1.82]; p-value = 0.053) in the Yoga group during the same time period. Conclusions: A structured Yoga intervention in the preoperative setting improved QOL, especially in the domains of sexual function, social, emotional and functional well-being as well as fatigue. Yoga positively modulates chronic low-grade inflammation which may impact tumor microenvironment. Yoga is feasible and safe and has promising benefits to physical, mental and emotional well-being. Health care providers can utilize the preoperative period as a window of opportunity for an intervention and a teaching moment for the patient to enhance their overall health. Further integrated large-scale research into molecular impact of yoga on markers of inflammation and immune function are essential. Source of Funding: This project was supported by the ThriveWell Cancer Foundation grant to Dharam Kaushik. The content is solely the responsibility of the authors and does not necessarily represent the official views of the ThriveWell Cancer Foundation. Citation Format: Dharam Kaushik, Pankil Shah, Kumar A. Pratap, Ian M. Thompson, Robert S. Svatek, Javier Hernandez, Ian Thompson, Ahmed Mansour, Richapriya Jha, Yang Xiaoyu, Deepak K. Pruthi, Hanzhang Wang, J. Ricardo Rivero, Nydia Darby, Ronald Rodriguez, Michael A. Liss. A randomized controlled trial of yoga in men with prostate cancer undergoing radical prostatectomy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-292.