scholarly journals The Novel BET Inhibitor PLX51107 Has In Vitro and In Vivo Activity Against Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3941-3941 ◽  
Author(s):  
Nicole R. Grieselhuber ◽  
Shaneice R. Mitchell ◽  
Shelley Orwick ◽  
Bonnie K. Harrington ◽  
Virginia M. Goettl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Term Survival ◽  
In Vivo Models ◽  
Bet Inhibitor ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) has very poor long-term survival with traditional therapies. AML has a diverse pathogenesis and likely represents multiple different diseases. Various epigenetic effector proteins are altered in AML by mutation, over-expression, or compartmental displacement and these changes maintain transcriptional programs important for leukemogenesis. The bromodomain and extra-terminal domain (BET) proteins, including BRD2, BRD3 and BRD4, play roles in many cellular functions important to leukemogenesis, such as super-enhancer function, transcriptional elongation, histone acetylation and cell cycle progression. In particular, AML cells depend on BRD4 for expression of the pro-survival proteins MYC and BCL2. BRD4 has therefore become an attractive target for novel therapeutics. PLX51107 is a novel BET inhibitor with a unique binding mode in the acetylated lysine binding pocket of BRD4 that differentiates it from other compounds under investigation. Our group has previously shown this compound to have antineoplastic activity in models of aggressive B cell malignancies. We have now investigated the anti-leukemic properties of PLX51107 in both in vitro and in vivo models of AML. Results: PLX51107 treatment potently reduced viability and proliferation of the human AML cell lines MV4-11, MOLM-13, OCI-AML3, and Kasumi-1, with IC50 of 0.17, 1.8, 0.2 and 0.2 μM, respectively. We then evaluated the in vitro activity of PLX51007 in primary human AML samples. PLX51107 inhibited the proliferation of primary human AML cells co-cultured with HS5 stromal cells. For nearly all samples tested (n=9), the IC50 of PLX51007 was less than 1 μM (average = 0.41 μM, range 0.039 - 1.5 μM). Notably, PLX51107 showed efficacy across a broad range of AML risk groups, including samples with adverse risk features such as 11q23 abnormalities and FLT3-ITD mutations. In comparison, for the same AML samples, the average IC50 for JQ1 was 0.71 μM (range 0.02 - 3.3 μM) and for cytarabine was 3.5 μM (range 0.33 to >10 μM). Furthermore, PLX51107 treatment reduced the clonogenicity of primary AML cells. Following incubation of AML cells in 1 μM PLX51107, there was significantly decreased colony formation (p<0.05) in drug-free, cytokine-supplemented methylcellulose media. We next examined the efficacy of PLX51107 in vivo, utilizing luciferase labeled MV4-11 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 47 days) compared to vehicle treated control animals (median 30 days, p< 0.001). Weekly measurement of bioluminescence showed decreased disease burden in PLX51107 treated mice. In addition, human peripheral blood CD45 / CD33 double positive cells were significantly decreased in treated animals. Histologic analysis conducted at day 16 showed decreased leukemic burden in the bone marrow of the PLX51107 treated animals. In addition, examination of tissues from moribund mice at time of euthanasia demonstrated fewer leukemia cells in the spleen, liver and bone marrow. Conclusions: Collectively, our results show pre-clinical activity of PLX51107 in AML, supporting further development of this compound in clinical trials for relapsed or refractory myeloid malignancies. We are currently working to define downstream targets of PLX51107 action and developing patient derived AML xenografts to further characterize the in vivo effects of PLX51107. Disclosures Walker: Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding.

scholarly journals Combination of BCL-2 Inhibition and Triptolide Causes Synthetic Lethality in Acute Myeloid Leukemia through Preventing Reciprocal Resistance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2083-2083
Author(s):  
Bing Xu ◽  
Yuanfei Shi ◽  
Long Liu ◽  
Bing Z Carter
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Synthetic Lethality ◽  
Leukemic Cells ◽  
Apoptotic Pathway ◽  
P53 Status ◽  
Acute Myeloid

BCL-2 inhibition exerts effective pro-apoptotic activities in acute myeloid leukemia (AML) but clinical efficacy as a monotherapy was limited in part due to the treatment-induced MCL-1 increase. Triptolide (TPL) exhibits anti-tumor activities in part by upregulating pro-apoptotic BCL-2 proteins and decreasing MCL-1 expression in various malignant cells. We hypothesized that combined BCL-2 inhibition and TPL exert synergistic anti-leukemia activities and prevent the resistance to BCL-2 inhibition in AML. We here report that TPL combined with BCL-2 inhibitor ABT-199 synergistically induced apoptosis in leukemic cells regardless of p53 status through activating the intrinsic mitochondrial apoptotic pathway in vitro. Although ABT-199 or TPL alone inhibited AML growth in vivo, the combination therapy demonstrated a significantly stronger anti-leukemic effect. Mechanistically, TPL significantly upregulated BH3 only proteins including PUMA, NOXA, BID and BIM and decreased MCL-1 but upregulated BCL-2 expression in both p53 wild type and p53 mutant AML cell lines, while the combination decreased both BCL-2 and MCL-1 and further increased BH3 only BCL-2 proteins. MCL-1 and BCL-2 increases associated with respective ABT-199 and TPL treatment and resistance were also observed in vivo. Significantly downregulating MCL-1 and elevating BH3 only proteins by TPL could not only potentially block MCL-1-mediated resistance but also enhance anti-leukemic efficacy of ABT-199. Conversely, BCL-2 inhibition counteracted the potential resistance of TPL mediated by upregulation of BCL-2. The combination further amplified the effect, which likely contributed to the synthetic lethality. This mutual blockade of potential resistance provides a rational basis for the promising clinical application of TPL and BCL-2 inhibition in AML independent of p53 status. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding.

scholarly journals Fc Receptor-Dependent Trogocytosis of CD39 Impacts Engraftment and Invasiveness of Acute Myeloid Leukemia Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3298-3298
Author(s):  
Lili Feng ◽  
Haohai Zhang ◽  
Paola de Andrade Mello ◽  
Dina Stroopinsky ◽  
Wenda Gao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bioluminescence Imaging ◽  
Lentiviral Vector ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Corresponding author: Dr. Simon. C. Robson ([email protected]). Introduction: CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the prototypic member of the GDA1-CD39 superfamily of ectonucleotidases and modulates purinergic signaling pathways. CD39 expression has been noted in human acute myeloid leukemia (AML) and likely contributes to chemoresistance [1]. Our study reported here elucidates the impact of Cd39 on engraftment and invasiveness of AML TIB-49 cells using an immunocompetent murine experimental model. Methods: Wild-type (WT) mice and Cd39 -/- mice on C57BL/6 background were bred at Beth Israel Deaconess Medical Center. The syngeneic murine AML cell line TIB-49 (Cd39 negative in vitro) was purchased from American Type Culture Collection. For bioluminescence imaging experiments, TIB-49 cells were transduced with luciferase/mCherry using a lentiviral vector. For AML model, mice were administered with 1×10 6 TIB-49-luciferase cells intravenously via tail vein injection. For chloroma model, mice were subcutaneously inoculated with 1×10 6 TIB-49 cells in the right flank. Bioluminescence imaging of TIB-49-luciferase bearing mice was conducted with the IVIS TM 50 Imaging System. Blood, spleen and bone marrow (BM) were also collected from TIB-49 bearing AML mice for FACS (fluorescence activated cell sorting) analysis. To explore Cd39 in TIB engraftment and invasiveness, TIB-49 cells were further transduced with a lentiviral vector overexpressing mCd39 with TdTomato. WT mice were intravenously inoculated with 1×10 6 of either TIB-49-TdTomato cells or TIB-49-mCd39-TdTomato cells, and the above read-outs were determined. To investigate the potential of CD39 as a therapeutic target, we engineered anti-mouse Cd39 antibodies (αCd39 mAb) with isotype selection and removal of fucose to further promote Fc receptor (FcR) interactions. Results: Bioluminescence imaging results indicated that TIB-49 engraftment was decreased in global Cd39 -/- mice with decreased disease burdens noted relative to WT (Figure 1A). FACS analysis of blood, spleen and BM-derived cells from TIB-49 bearing AML-model mice (day 31) confirmed higher engraftment of TIB-49 cells (TdTomato+) at all sites in WT compared to Cd39 -/- mice (Figure 1B). TIB-49 cells did not express Cd39 in vitro, but TIB-49 cells harvested from spleen and BM of WT but not Cd39 -/- mice displayed high levels of Cd39. This indicated TIB-49 cells acquired Cd39 from host cells, in a process of antibody-independent trogocytosis (Figure 1C), as RT-PCR did not detect Cd39 mRNA expression in TIB-49 cells in vivo. Additionally, circulating TIB-49 cells from the blood of WT mice were Cd39 negative (Figure 1C), suggesting a role for the tumor microenvironment in mediating trogocytosis. TIB-49 cells expressing host Cd39 in WT mice spleen and BM lost Cd39 after being exposed to αCd39 mAb treatment. Cd39 translocated from TIB-49 cells to effector cells, at least in part, dependent on FcR mediated trogocytosis (Figure 1D). When Cd39 was overexpressed on TIB-49 cells (TIB-49-mCd39-TdTomato), the engraftment was boosted in WT mice in vivo when compared to TIB-49-TdTomato cells (day 19, Figure 1E) with higher levels of Cd39 expression than that observed on TIB-49-TdTomato cells in spleen and BM (day 26) (Figure 1F). Moreover, TIB-49-mCd39-TdTomato bearing mice displayed shorter survival times, when compared with TIB-49-TdTomato bearing AML mice (Figure 1G). The αCd39 mAb monotherapy had no effect on TIB-49 chloroma model growth. However, pretreatment with αCd39 mAb effectively boosted daunorubicin chemotherapeutic effects in vivo (Figure 1H and 1I). Conclusions: Our study suggests bidirectional trogocytosis between TIB-49 AML and host immune cells, which is further modulated by FcR interaction. Re-distribution of Cd39 from host to TIB-49 cells or induced high level expression contributes to engraftment and invasiveness, resulting in decreased survival. Targeting CD39 is a potential therapeutic approach, operational not only by boosting chemosensitivity but furthering anti-leukemic effects in experimental models. Disclosures: No relevant conflicts of interest to declare. References: [1] Nesrine Aroua, Emeline Boet, Margherita Ghisi, et al. Extracellular ATP and CD39 Activate cAMP-Mediated Mitochondrial Stress Response to Promote Cytarabine Resistance in Acute Myeloid Leukemia. Cancer Discov. 2020. Figure 1 Figure 1. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: 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; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

scholarly journals The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1663 ◽  
Author(s):  
Arne Velthaus ◽  
Kerstin Cornils ◽  
Jan K. Hennigs ◽  
Saskia Grüb ◽  
Hauke Stamm ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Actin Binding ◽  
Bone Marrow Niche ◽  
Actin Binding Protein ◽  
Acute Myeloid

Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.

Disulfiram, an clinically used anti-alcoholism drug has the potential to target acute myeloid leukemia cells in a copper-dependent manner in vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5040-5040
Author(s):  
Bing Xu ◽  
Rongwei Li ◽  
Huijuan Dong ◽  
Feili Chen ◽  
Yuejian Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Specific Antigen ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Control Group ◽  
Acute Myeloid

Abstract Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p < 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti-Leukemic Activity of Daratumumab in Acute Myeloid Leukemia Cells and Patient-Derived Xenografts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2312-2312 ◽  
Author(s):  
Cedric Dos Santos ◽  
Shan Xiaochuan ◽  
Zhou Chenghui ◽  
Georges Habineza Ndikuyeze ◽  
Joshua Glover ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Killing ◽  
Nsg Mice ◽  
Cd38 Expression ◽  
Acute Myeloid

Abstract Daratumumab is a human antibody that binds to CD38 on the cell surface and induces cell killing by multiple mechanisms including complement mediated cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and apoptosis. In pre-clinical and clinical studies, daratumumab has been shown to effectively kill multiple myeloma (MM) cells and to enhance the potency of other treatments against MM. The purpose of the study was to investigate in vitro and in vivo efficacy of daratumumab against 9 acute myeloid leukemia (AML) cell lines and patient-derived samples. First, we evaluated the expression of CD38, complement inhibitory proteins (CIP) CD46, CD55, CD59, and FcgR1 (CD64) on AML cell lines (n=9), AML patient cells (n=10) and healthy donor bone marrow using flow cytometry. CD38 enumeration showed a substantial variation between cell lines (12,827±19, 320 molecules/cell) and between AML patients (11,560±8, 175 molecules/cell), while CD38 expression was more consistent in bone marrow (BM) from healthy donors (1,176±355 molecules/cell). The daratumumab-induced apoptosis observed in cell lines (MOLM-13, MOLM-16, MV-4-11, NB4) in vitro was not correlated with CD38 expression levels. Daratumumab induced minimal ADCC (5-20%) and low levels of (2-5%) CDC mediated cell killing in 6 AML cell lines tested. We did not observe a direct correlation between CD38 expression and ADCC, CDC, nor between CDC and CIP expression. Interestingly, treatment of two human Acute Promyelocytic Leukemia (M3) cell lines HL-60 and NB-4 with all-trans retinoic acid (ATRA) induced a 10-30-fold increase in CD38 expression, suggesting that ATRA could be used in combination with daratumumab. While we, and others, have shown that pre-incubation of primary AML cells with anti-CD38 antibodies inhibits engraftment in NSG mice, we aimed at evaluating the anti-leukemic activity of daratumumab in a therapeutic xenograft model using 3 different AML patients. NSG mice (10/group/patient) were transplanted with T cell-depleted AML cells and BM aspirates were collected 4-6 weeks later to assess leukemia burden in each mouse prior to treatment. Animals were untreated (Ctrl) or received daratumumab (10 mg/kg), or IgG1 isotype once a week for five weeks. We assessed AML burden (% huCD45+ CD33+) in BM, spleen (SPL) and peripheral blood (PB) within 5 days after the last treatment. First, we evaluated an AML (#3406, FLT3-ITD, see figure) with high expression of CD38 (13,445 molecules/cell) and low CD64 (489/cell) was evaluated. Daratumumab significantly reduced leukemia burden in SPL and PB, but had no effect in BM. The same daratumumab-induced reduction in peripheral blasts and lack of effect in BM was observed in 2 other AML patient xenografts (#7577, M1 IDH mutant/FLT3-ITD with 6,529 CD38 molecules/cell; #8096, M2 with 335 CD38 molecules/cell). Interestingly, we observed that daratumumab treatment led to a drastic reduction in CD38 surface expression in AML blasts including in BM, indicating that daratumumab efficiently targeted CD38 in bone marrow blasts. Our results suggest that the bone marrow microenvironment can impair the anti-leukemic activity of daratumumab observed in other tissues. Ongoing xenograft studies are testing whether induction with chemotherapy (Ara-C+doxorubicin), or with other agents disrupting the bone marrow microenvironment, can enhance the anti-leukemic activity of daratumumab. Figure 1: Effect of daratumumab treatment on AML 3406 leukemia burden: Figure 1:. Effect of daratumumab treatment on AML 3406 leukemia burden: Disclosures Dos Santos: Janssen R&D: Research Funding. Xiaochuan:Janssen R&D: Research Funding. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. Danet-Desnoyers:Janssen R&D: Research Funding.

scholarly journals A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2709-2709
Author(s):  
Haydar Celik ◽  
Katherine E. Lindblad ◽  
Bogdan Popescu ◽  
Giovanna Fantoni ◽  
Gege Gui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Serum Samples ◽  
Advisory Committees ◽  
Extracellular Compartment ◽  
Acute Myeloid

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe & Dohme: Research Funding.

p38MAPK Inhibition Blocks Inflammatory Signaling in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2603-2603 ◽  
Author(s):  
Alyssa Carey ◽  
Swati Garg ◽  
Megan M Cleary ◽  
David K Edwards ◽  
Marc Loriaux ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hematologic Malignancies ◽  
Sensitivity Profile ◽  
Acute Myeloid

Abstract Background: p38 mitogen-activated protein kinase (p38MAPK) is activated by various pro-inflammatory and stress-related stimuli, and has been an attractive therapeutic target for autoimmune diseases. p38MAPK (hereafter referred to as p38) signaling is also involved in cell proliferation, differentiation, apoptosis, and invasion, suggesting that it may be a potential therapeutic target for cancer. We found that inflammatory cytokines, including interleukin-1 (IL-1), promote growth and survival of more than half of the acute myeloid leukemia (AML) patient samples we tested. Since p38 is a downstream mediator of inflammatory pathways, we hypothesized that targeting p38 might be an effective therapeutic strategy in AML and other hematologic malignancies. To test this hypothesis, we evaluated the effectiveness of three p38 inhibitors using in vitro studies in primary AML patient samples. We found that targeting p38 blocks IL-1-activated extrinsic signaling and is a critical therapeutic target in a large subset of AML patients. Methods: We screened ~1000 primary leukemia patient samples for sensitivity to p38 inhibition using varying concentrations of doramapimod (BIRB 796) in a cell growth assay. We compared the sensitivity profile of doramapimod with 2 other small-molecule p38 inhibitors currently in clinical trials: ARRY 614, a dual p38/Tie2 inhibitor, and ralimetinib, which blocks activation of p38 by its substrate MK2. We determined cell viability, survival, and downstream signaling in the presence of 10 ng/ml IL-1α or IL-1β. Patient samples with IC50 < 1000nM were considered drug responsive. Results: In our patient population, we observed response rates of 31% in AML (109/350), 27% in myelodysplastic syndromes (MDS; 25/93), 19% in myeloproliferative neoplasms (23/123), 13% in mature B-cell neoplasms (30/232), and 10% in precursor lymphoid neoplasms (19/182). Focusing on AML, we compared the sensitivity profile of doramapimod with two other small-molecule p38 inhibitors, ralimetinib and ARRY 614. These inhibitors showed strikingly similar sensitivity profiles to doramapimod when tested in an additional 25 primary AML samples, with ~25% responsive and median IC50 of 11 nM for ARRY 614 (range: 7-650nM), 105 nM for ralimetinib (range: 7-850nM), and 18 nM for BIRB 796 (range: 13-40nM). Because IL-1 is known to stimulate p38 signaling, we compared the response rates for these three p38 inhibitors with or without IL-1 in a dose-response study. IL-1 increased the percent of AML samples responding to p38 inhibition from 25% to 60%, indicating a potentially important role of extrinsic inflammatory stimuli in p38 inhibitor sensitivity. Consistent with this all three p38 inhibitors were similarly effective in blocking the growth of primary AML CD34+ progenitors, suggesting that targeting p38 might reduce early progenitor AML cells. Further, we compared doramapimod, ralimetinib, and ARRY 614 for their ability to inhibit p38 phosphorylation in primary AML samples using flow cytometry and immunoblot analysis; all three inhibitors blocked p38 pathway activation in AML cells. Notably, in clinical studies of ARRY 614 in MDS patients, preliminary biomarker analyses demonstrated persistent inhibition of phospho-p38 in the bone marrow during the treatment. Also, consistent with functional inhibition of p38, there was a profound decrease in plasma cytokine concentrations, most significantly IL-1, during ARRY 614 treatment. In 250 primary AML samples, we observed no correlation between BIRB 796 sensitivity in vitro and clinical metrics such as white blood cell count, blast percentage in peripheral blood or bone marrow, karyotype, or tumor genotype. This suggests that IL-1 and p38 activation might be independent biomarkers of drug sensitivity. Conclusions: These data underscore the importance of the p38MAPK pathway in the pathobiology of AML and provide strong preclinical evidence to support p38MAPK as a therapeutic target. Targeting p38MAPK might also block tumor-extrinsic signaling, as indicated by IL-1-activated signaling. That all three p38MAPK inhibitors showed comparable sensitivity profiles holds promise for ARRY614, which showed the lowest median IC50 and is currently in clinical development. In addition, with further study these findings may be extended to hematologic malignancies other than AML. Disclosures Winski: Array BioPharma Inc.: Employment. Cable:Array BioPharma Inc.: Employment. Tyner:Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding. Agarwal:CTI BioPharma: Research Funding.

HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1456-1466 ◽  
Author(s):  
Neal A. Fischbach ◽  
Sofia Rozenfeld ◽  
Weifang Shen ◽  
Stephen Fong ◽  
Daniel Chrobak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hox Genes ◽  
Cooperative Interaction ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Acute Myeloid

AbstractThe HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors, and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis, we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner, pre–B-cell leukemia transcription factor-1 (PBX1). Additionally, we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo, HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4, a region frequently deleted in HOXA9-induced AMLs. In vitro, HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1.

scholarly journals Using an in-Vivo Degron-Based Approach to Interrogate Dependencies of Serially Acquired Mutations - Including DNMT3a-R882 and NPM1c - in Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Andrew A Guirguis ◽  
Alexander Tepper ◽  
Max Jan ◽  
Quinlan Sievers ◽  
Benjamin L. Ebert
Keyword(s):  
Acute Myeloid Leukemia ◽  
Targeted Therapies ◽  
Myeloid Leukemia ◽  
Single Amino Acid ◽  
Triple Mutant ◽  
Term Survival ◽  
Leukemia Development ◽  
Acute Myeloid

While a number of targeted therapies have recently become available in the treatment of acute myeloid leukemia (AML), it remains to be determined whether early acquired mutations are essential for ongoing leukemia maintenance in the face of sequential acquisition of additional mutations. In some cases, our ability to answer this question is hampered by the lack of targeted therapies to a number of these mutations. Of all AML mutations, those involving DNMT3a and NPM1c are some of the most frequent - often acquired before the final transforming event. Here we report the development of an in-vivo degron-based approach to interrogate the requirement for DNMT3a-R882 and NPM1c after leukemia development. Using an IKZF-based degron system offers us the opportunity to address the ongoing requirement of particular oncogenes for AML maintenance - by way of a conditional system that can be rapidly modulated. Thalidomide and its related analogues act by recruiting various substrates to the CRL4CRBN E3-ubiquitin ligase complex mediating subsequent ubiquitination and proteasomal degradation. In a majority of cases - the basis of this degradation has been determined to be the result of recognition and binding to a degron motif within C2H2 zinc fingers. Using more detailed analysis with single amino-acid resolution, we have engineered 'degrons' sensitive or resistant to degradation by thalidomide and its related analogues. In-vivo degradation using pomalidomide revealed a dependence of GMP cells on the presence of NPM1c but not DNMT3a R882. Using our cereblon (CRBN) knock-in mice engineered to mediate sensitivity to these compounds, we generated triple-mutant acute-myeloid leukemia - sequentially introducing DNMT3a-R882, NPM1c and N-RAS G12D mutations whilst altering the site of the degron tag in each case. Degradation-resistant degron constructs were used as a control in each case. Findings were subsequently correlated and validated with in-vitro treatments of triple mutant cells. RNA-seq was also performed after both in-vitro and in-vivo treatments to further delineate acute transcriptional changes in the absence of NPM1c - highlighting a number of changes including those within the Hox family of genes Together, these findings suggest triple mutant leukemia remains dependent on the leukemia-inducing mutation of NPM1c. In contrast, whilst epigenetic alterations induced by DNMT3a mutations are essential for leukemia development - these do not appear to be essential for ongoing leukemia maintenance. Further work is currently underway to determine how modulating NPM1c impacts long-term survival from leukemia and whether the epigenetic changes associated with DNMT3a R882 mutations are indeed reversible in the context of leukemia. This work highlights an approach to interrogate a protein of interest in-vivo using a conditional system that is reversible, rapidly acting and that uses a series of FDA-approved compounds with known kinetics. We envisage this will be generalisable as a tool to be used in other similar contexts. Disclosures No relevant conflicts of interest to declare.

scholarly journals Prolyl Hydroxylase 3 (Phd3) Is a Therapeutic Target in Isocitrate Dehydrogenase 1 (IDH1) Mutated Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 759-759
Author(s):  
Anuhar Chaturvedi ◽  
Michelle Maria Araujo Cruz ◽  
Ramya Goparaju ◽  
Razif Gabdoulline ◽  
Renate Schottmann ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Pdx Model ◽  
Immunocompromised Mice ◽  
Mutant Idh1 ◽  
Acute Myeloid

Abstract Background: About 40% of IDH1 mutated (IDH1mut) acute myeloid leukemia (AML) patients respond to IDH1 inhibitors with a median duration of response of 8.2 months. A better understanding of the biology of IDH1mut leukemia may further improve the treatment of these patients. IDH1mut produces R-2-hydroxyglutarate (R-2HG), which activates PHD1 and PHD2 but have negligible effects on PHD3. In the present study we assessed whether PHD3 plays a role in the pathogenesis of IDH1 mutated leukemia and can be targeted in a patient-derived xenograft (PDX) model of IDH1 mutated AML. Methods: Bone marrow cells from Phdwt and Phd3ko mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt) and IDH1mut respectively, were constitutively expressed. The effects on cell proliferation, apoptosis and colony formation were evaluated in vitro, whereas the leukemic potential was evaluated in vivo by transplantation in syngeneic mice. To show that PHD3 is a therapeutic target, either IDH1mut cells from AML patients were transduced with shRNA against PHD3 and transplanted in immunocompromised mice, or leukemic cells from an AML patient with mutated IDH1 were xenografted in immunocompromised mice and treated with the PHD inhibitor molidustat. Results: In in-vitro functional assays loss of Phd3 specifically impaired proliferation, apoptosis and clonogenic capacity of HoxA9-IDH1mut but not HoxA9-IDH1wt cells. Likewise, in mouse transplantation assays, loss of Phd3 eliminated HoxA9-IDH1mut induced leukemia. However, Phd3 was dispensable to the engraftment and proliferation of HoxA9-IDH1wt cells. Additionally, the IDH1-independent model of MN1-induced leukemia remained unaltered in the absence of Phd3, indicating the specificity of the role of Phd3 in mutant IDH1-induced transformation. To identify molecular pathways that might explain in vitro and in vivo phenotypes gene expression profiling was performed. Immune and stress-response pathways as well as metabolism-related genes were most prominently dysregulated in Phd3ko IDH1-mutant cells. Analysis of dysregulated transcription factors by gene set enrichment analysis revealed a depletion of key oncogenic transcription factors (Myc, Rb, Stk33, and Rps14) in Phd3ko IDH1mut cells compared to Phd3ko IDH1wt cells. To study if IDH1mut signals to Phd3 through R-2HG, we transduced Phd3kocells, with a splice variant of mutant IDH1, which does not produce R-2HG but causes leukemia in mice with similar kinetics as in mice with the full-length IDH1 mutant protein. Interestingly, loss of Phd3 also eliminated leukemia in these mice, which demonstrates that mutant IDH1 signals through Phd3 independently of R-2HG. To study the functional relevance of PHD3 inhibition in patients, cells from an IDH1 mutated AML patient were transduced with an shRNA against PHD3 and were transplanted in immunodeficient NSG mice. Inhibition of PHD3 depleted human AML cells in the IDH1-mutated PDX model. Moreover, the PHD inhibitor molidustat was 50-fold more active in IDH1mut (80 nM) compared to IDH1wt AML patient cells (4000 nM) in colony-forming unit assays. In a xenograft model of IDH1 mutated AML, molidustat significantly prolonged survival compared to control-treated mice (P<.001). Conclusion: We demonstrate that the leukemogenic activity of the mutant IDH1 protein depends on PHD3 independently of R-2HG. We identified inhibition of PHD3 as a novel therapeutic strategy in IDH1 mutated AML. Since PHD3 can be targeted pharmacologically, combinatorial treatment of PHD3 and IDH1 inhibitors is warranted to improve eradication of leukemic stem cells in IDH1 mutated AML. #AC and MMAC share first authorship Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Karyopharm: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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