Targeting Aurora Kinase B with AZD2811 Enhances Venetoclax Activity in TP53-Mutant AML

Background: Recent data suggests that older patients with acute myeloid leukemia (AML) have a higher frequency of poor risk TP53 mutations (TP53mut) than previously suspected. Patients with TP53mut have a very poor prognosis, making this AML sub-group an area of high unmet therapeutic need. Although recent clinical trials suggest promising response rates for the BCL-2 inhibitor venetoclax (Ven) in combination with DNA methyltransferase inhibitors (DNMTi) or low-dose cytarabine, survival outcomes remain poor among patients with TP53mut (Strickland et al, EHA 2018). Methods and Results: To identify novel therapies effective against TP53mut AML, isogenic TP53 knockout cells (TP53 KO) were generated by CRISPR/Cas9 in OCI-AML3, MV4;11 and MOLM-13 human AML cell lines. In a drug screen of 50 compounds, we identified AZD2811, an aurora kinase B inhibitor, which potently killed AML cells independently of TP53 genotype (Figure 1). We hypothesised that AZD2811 would synergise with Ven to overcome venetoclax-resistance in TP53-mutant AML. Consistent with this, AZD2811 and venetoclax showed strong synergy (Loewe score <4) in 6 AML cell lines independent of TP53 genotype, and combined treatment displayed highly efficacious activity in both TP53 KO cells and wild type control cells in vitro (Figure 2). Immunoblot of treated cells revealed that AZD2811 depleted MCL-1 expression in the majority of cases, a known cause of venetoclax resistance. AZD2811 was also active in killing Bax/Bak knockout cells, suggesting that the mechanism of AZD2811 may not be limited to the intrinsic apoptosis pathway. Using a competitive growth assay incorporating a fluorescent reporter for TP53 KO cells, we showed the relative resistance of TP53 defective cells to Ven and decitabine (DEC) alone, as well as Ven + DEC, compared to wild type cells. In contrast, TP53 KO cells showed no competitive growth advantage when exposed to an initially sub-lethal dose of AZD2811 (10 nM), compared to TP53 WT cells. Interestingly, continuous exposure to AZD2811 resulted in a catastrophic collapse in cell viability on day 10, unlike comparator anti-leukemic agents (Figure 4). In vivo efficacy for nanoparticle formulated AZD2811 (NP) was observed in SCID mice xenografted with HL-60 cells (known to be TP53 mutant), where combined treatment of AZD2811NP and Ven significantly prolonged animal survival (Figure 3). Remarkably, combined AZD2811NP and Ven treatment was more effective than Ven + azacitidine treatment in vivo. Patient-derived xenograft (PDX) models to verify the activity of AZD2811NP + venetoclax against primary AML cases with TP53 mutations will be presented at the meeting. Conclusions: We report for the first time that AZD2811NP can overcome venetoclax resistance in TP53-mutant AML in vitro and in vivo. These findings therefore, support the clinical investigation of combined aurora kinase and BCL-2 targeting in the clinic for patients with TP53-mutant AML, who currently lack effective treatment options. Disclosures Urosevic: AstraZeneca: Employment. Polanska:AstraZeneca: Employment. Cosaert:AstraZeneca: Employment. Pease:AstraZeneca: Employment, Equity Ownership. Travers:AstraZeneca: Employment. Wei:Pfizer: Honoraria; Janssen: Honoraria; Walter and Eliza Hall Institute: Other: former employee, Patents & Royalties: receives a fraction of its royalty stream related to venetoclax; AbbVie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Macrogenics: Honoraria; Astellas: former employee, Honoraria; Genentech: Honoraria; Servier: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

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Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Blood ◽

10.1182/blood-2021-149442 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 248-248

Author(s):

Alice Bonato ◽

Riccardo Bomben ◽

Supriya Chakraborty ◽

Giulia Felician ◽

Claudio Martines ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Pi3k Inhibitor ◽

Leukemia Cells ◽

Wild Type ◽

Clonal Composition

Abstract Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P<0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P<0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P<0.001, with 1.0 μM IBR: 28% vs 53%, P<0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with >10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

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Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-145956 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 615-615

Author(s):

Yuxuan Liu ◽

Lucille Stuani ◽

Dorra Jedoui ◽

Milton Merchant ◽

Astraea Jager ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Bcr Signaling ◽

Proliferation In Vitro

Abstract Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

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Use of Medium Potency Statins Is Associated with Improved Outcomes after Frontline RCHOP in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Blood ◽

10.1182/blood-2020-136010 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 45-45

Author(s):

Sushanth Gouni ◽

Paolo Strati ◽

Jason Westin ◽

Loretta J. Nastoupil ◽

Raphael E Steiner ◽

...

Keyword(s):

Cell Lines ◽

Research Funding ◽

Response Assessment ◽

Cholesterol Content ◽

In Vivo Studies ◽

High Cholesterol ◽

Improved Outcomes ◽

Significant Difference

Background: Pre-clinical studies show that statins may improve the efficacy of chemoimmunotherapy in patients with DLBCL, through interference with cell membrane-initiated signaling pathways. Clinical retrospective studies, however, yield conflicting data, due to heterogeneous properties of statins, including potency and hydrophilicity. Methods: This is a retrospective analysis of patients with previously untreated, advanced stage DLBCL, non-double hit, treated with frontline R-CHOP between 01/01/2000 and 09/01/2019 (data cut-off 04/15/2020) at MD Anderson Cancer Center, and for whom data regarding statin use at time of initiation of treatment were available. Lugano 2014 response criteria were applied retrospectively for response assessment. Cellular cholesterol levels were analyzed in 6 DLBCL cell lines using an Amplex red fluorometric assay. A doxorubicin (DXR)-resistant cell line was generated exposing SUDHL4 cells to escalating doses of DXR; a DXR-resistant DLBCL patient-derived xenograft (PDX) model was established through serial transplantation and exposure to DXR. Results: 271 patients were included in the analysis, 182 (67%) were older than 60 years, 134 (49%) were male, 212 (72%) had stage IV disease, and 217 (80%) had an IPI score > 3; upon pathological review, 38 (36%) cases were non-GCB type, and 18 (28%) were double-expressors; 214 (79%) were able to complete all planned 6 cycles of RCHOP. Seventy-nine (29%) patients received statins at time of initiation of chemoimmunotherapy: 15 patients received low potency statin, 51 medium and 13 high; 18 patients received hydrophilic statins and 61 lipophilic. Patients receiving statins were significantly older as compared to patients who did not (p<0.001); no other significant difference in baseline characteristics was observed when comparing the 2 groups. Overall, 265 out of 271 patients were evaluable for response, as 6 stopped treatment because of toxicity before first response assessment. Among these, ORR was 95% (252/265) and CR rate was 62% (165/265). ORR rate was identical in patients who were treated with statin and those who did not (95% both, p=1). After a median follow-up of 77 months (95% CI, 70-84 months), 119 patients progressed/died, median PFS was not reached and 6-year PFS was 57%. 6-year PFS rate according to statin intensity was: 48% (low), 72% (medium), 57% (high). PFS. 6-year PFS rate was 64% for hydrophilic and 72% for lipophilic statins. Patients treated with statins had a trend for longer PFS (p=0.06), significantly longer for patients receiving medium potency statins (p=0.04). No significant difference in PFS was observed when comparing patients treated with lipophilic statins to all others (not reached vs 84 months, p=0.22). To confirm these clinical data, in-vitro and in-vivo studies were performed. Six cell lines were tested: 4 with high cholesterol content (SUDHL4, HBL1, HT, and U2932; 5.0-8.0 µg/mg protein), and 2 with low cholesterol content (DOHH2 and OCI-LY19; 1.5-2.0 µg/mg protein); the latter showed the highest sensitivity to DXR-mediated killing. The combination of lovastatin and DXR (10nM) was tested in all 4 cell lines with high cholesterol content, resulting in more cell death than either treatment alone. Lovastatin (at the nanomolar range) resensitized DXR-resistant SUDHL4 cells to DXR. Finally, in a DXR-resistant PDX model, the combination of lovastatin and DXR resulted in delayed tumor growth as compared to chemotherapy alone. Conclusions: Use of medium potency statins is associated with improved outcomes after frontline RCHOP in patients with DLBCL. This was further confirmed in functional in-vitro and in-vivo studies. Future interventional studies, aimed at improving outcomes in these patients using this novel combination, are warranted. Disclosures Westin: Amgen: Consultancy; 47: Research Funding; Kite: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Morphosys: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Curis: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding. Nastoupil:Gamida Cell: Honoraria; Merck: Research Funding; TG Therapeutics: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; LAM Therapeutics: Research Funding; Novartis: Honoraria, Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Gilead/KITE: Honoraria. Neelapu:Bristol-Myers Squibb: Other: personal fees, Research Funding; Merck: Other: personal fees, Research Funding; Kite, a Gilead Company: Other: personal fees, Research Funding; Pfizer: Other: personal fees; Celgene: Other: personal fees, Research Funding; Novartis: Other: personal fees; Karus Therapeutics: Research Funding; N/A: Other; Takeda Pharmaceuticals: Patents & Royalties; Acerta: Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Precision Biosciences: Other: personal fees, Research Funding; Legend Biotech: Other; Adicet Bio: Other; Allogene Therapeutics: Other: personal fees, Research Funding; Cell Medica/Kuur: Other: personal fees; Calibr: Other; Incyte: Other: personal fees; Unum Therapeutics: Other, Research Funding. Landgraf:NCI/NIH: Research Funding. Vega:NCI: Research Funding.

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Abstract 470: Recording Cell Migration Dynamics in Mouse Tissues With Cells Secreting Fluorescence Protein-tagged Collagen

Circulation Research ◽

10.1161/res.127.suppl_1.470 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Zhongming Chen

Keyword(s):

Cell Migration ◽

Cell Lines ◽

Wild Type Mouse ◽

Cardiac Progenitor Cells ◽

Wild Type ◽

Mouse Tissues ◽

Migration Dynamics ◽

Fluorescence Protein

Background: Cell migration is an important step involved in heart regeneration and many cardiovascular diseases. However, cell migration dynamics in vivo is poorly understood due to the challenges from mammal hearts, which are opaque and fast beating, and thus individual cardiac cells cannot be imaged or tracked. Aims: In this study, cell migration dynamics in the heart is recorded with a novel strategy, in which fluorescence protein-tagged collagen is secreted from cells and deposited into extracellular matrix, forming visible trails when cells are moving in tissues. As a proof-of-concept, transplanted migration dynamics of cardiac progenitor cells in mouse hearts were investaged. Methods: Stable cell lines expressing mCherry-tagged type I collagen were generated from isolated cardiac progenitor cells, ABCG2 + CD45 - CD31 - cells (side populations), or c-kit + CD45 - CD31 - cells (c-kit + CPCs). The cell migration dynamics were monitored and measured based on the cell trails after cell transplantation into mouse tissues. Results: The stable cell lines form red cell trails both in vitro and in vivo (Fig. 1A & 1B, Green: GFP; Red: mCherry-collagen I, Blue: DAPI, bar: 50 microns). In culture dishes, the cells form visible cell trails of fluorescence protein. The cell moving directions are random, with a speed of 288 +/- 79 microns/day (side populations, n=3) or 143 +/-37 microns/day (c-kit + CPCs, n=3). After transplantation into wild-type mouse hearts, the cells form highly tortuous trails along the gaps between the heart muscle fibers. Angle between a cell trail and a muscle fiber is 16+/-16 degree (n=3). Side populations migrate twice as fast as c-kit+ CPCs in the heart (16.0 +/-8.7 microns/day vs. 8.1+/-0.0 microns/day, n=3, respectively), 18 time slower than the respective speeds in vitro . Additionally, side populations migrate significantly faster in the heart than in the skeletal muscles (26.4+/-5.8 microns/day, n=3). The side populations move significantly faster in immunodeficient mouse hearts (36.7+/-13.3 microns/day, n=3, typically used for studying cell therapies) than in wild-type mouse hearts. Conclusion: For the first time, cell migration dynamics in living hearts is monitored and examined with genetically modified cell lines. This study may greatly advance the fields of cardiovascular biology.

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Radiosensitisation of Hepatocellular Carcinoma Cells by Vandetanib

Cancers ◽

10.3390/cancers12071878 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1878 ◽

Cited By ~ 2

Author(s):

Sami Znati ◽

Rebecca Carter ◽

Marcos Vasquez ◽

Adam Westhorpe ◽

Hassan Shahbakhti ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Combination Therapy ◽

Cell Lines ◽

Radiation Treatment ◽

Combined Treatment ◽

New Combination ◽

Migration And Invasion ◽

Syngeneic Mouse Model

Hepatocellular Carcinoma (HCC) is increasing in incidence worldwide and requires new approaches to therapy. The combination of anti-angiogenic drug therapy and radiotherapy is one promising new approach. The anti-angiogenic drug vandetanib is a tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and RET proto-oncogene with radio-enhancement potential. To explore the benefit of combined vandetanib and radiotherapy treatment for HCC, we studied outcomes following combined treatment in pre-clinical models. Methods: Vandetanib and radiation treatment were combined in HCC cell lines grown in vitro and in vivo. In addition to 2D migration and clonogenic assays, the combination was studied in 3D spheroids and a syngeneic mouse model of HCC. Results: Vandetanib IC 50 s were measured in 20 cell lines and the drug was found to significantly enhance radiation cell kill and to inhibit both cell migration and invasion in vitro. In vivo, combination therapy significantly reduced cancer growth and improved overall survival, an effect that persisted for the duration of vandetanib treatment. Conclusion: In 2D and 3D studies in vitro and in a syngeneic model in vivo, the combination of vandetanib plus radiotherapy was more efficacious than either treatment alone. This new combination therapy for HCC merits evaluation in clinical trials.

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CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.641.641 ◽

2004 ◽

Vol 104 (11) ◽

pp. 641-641 ◽

Cited By ~ 1

Author(s):

Suzanne Trudel ◽

Zhi Hua Li ◽

Ellen Wei ◽

Marion Wiesmann ◽

Katherine Rendahl ◽

...

Keyword(s):

Multiple Myeloma ◽

Mouse Model ◽

Tyrosine Kinase ◽

Cell Lines ◽

Small Molecule ◽

Kinase Inhibitor ◽

Wild Type ◽

Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

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Recombinant Human Erythropoietin (rHuEPO) In Combination with Chemotherapy Increases Breast Cancer Metastasis In Pre-Clinical Mouse Models

Blood ◽

10.1182/blood.v116.21.3345.3345 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3345-3345

Author(s):

Anargyros Xenocostas ◽

Benjamin D Hedley ◽

Jenny E Chu ◽

D. George Ormond ◽

Michel Beausoleil ◽

...

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Research Funding ◽

Breast Cancer Cells ◽

Cancer Metastasis ◽

Breast Cancer Metastasis ◽

In Vivo Studies ◽

Metastatic Process

Abstract Abstract 3345 Background: Erythropoietin (EPO) is a key regulator of erythropoiesis, and has been shown to stimulate growth, maintain viability, and promote differentiation of red blood cell precursors. The EPO receptor (EPO-R) is expressed by erythroid cells and by several non-hematopoietic cell types including various neoplastic cells. Erythropoiesis-stimulating agents (ESAs) are used clinically for the treatment of chemotherapy-induced anemia. The results of some recent randomized clinical trials have reported an increased incidence in adverse events and reduced survival in ESA-treated metastatic breast cancer patients receiving chemotherapy, potentially related to EPO-induced cancer progression. These results have raised concerns over ESA treatment in metastatic cancer patients. However, very little pre-clinical data is available regarding the impact of EPO on breast cancer metastasis. The goal of the current study was therefore to determine if EPO can influence the malignant behavior of breast cancer cells and/or influence the metastatic process. Methods: MDA-MB-468, MDA-MB-231, MDA-MB-435, and 4T-1 breast cancer cell lines were treated with recombinant human EPO (rHuEPO; 10 U/ml) or control media and screened for EPO-R mRNA expression levels by RT-PCR, and for EPO-R protein expression by Western blot and flow cytometry. MDA-MB-231 (231) and MDA-MB-435 (435) cell lines were used for functional assays in vitro and in vivo. Untreated or rHuEPO treated cells were grown in 2D and 3D in vitro systems (standard tissue culture plates and 0.6% soft agar, respectively) to determine if rHuEPO influenced growth. In vitro cell survival was also assessed in response to treatment with rHuEPO in the presence or absence of paclitaxel chemotherapy (10mg/ml), radiation (10G), or hypoxic conditions (1% O2). Following mammary fat pad injection, in vivo effects of rHuEPO (300U/kg) alone or in combination with paclitaxel treatment (10mg/kg) were assessed in mouse models of tumorigenicity and spontaneous metastasis. Results: Expression analysis of EPO-R mRNA and protein revealed a large variation in levels across different cell lines. The majority of cell lines did not express cell surface EPO-R by flow cytometry, although two cell lines (231 and 435) did show weak expression of EPO-R mRNA, with only the 231 cell line showing EPO-R expression by Western blot. In vitro, a small protective effect from rHuEPO on radiation-treated 435 cells was seen (p<0.05); however, rHuEPO treatment alone or combined with chemotherapy or hypoxia did not cause a significant increase in cell survival relative to untreated controls cells. In contrast, in vivo studies demonstrated that rHuEPO increased the incidence and burden of lung metastases in immunocompromised mice injected with 231 or 435 cells and treated with paclitaxel relative to mice treated with paclitaxel alone (p<0.05). Conclusions: The lack of an in vitro effect of rHuEPO highlights the importance of in vivo studies to delineate the effects of EPO on the metastatic process. Our novel findings demonstrate that rHuEPO can reduce the efficacy of chemotherapy in the metastatic setting in vivo, and in some cases enhance the inherent metastatic growth potential of human breast cancer cells. This work was supported by funding from the London Regional Cancer Program and Janssen Ortho Canada Disclosures: Xenocostas: Janssen Ortho: Consultancy, Honoraria, Research Funding. Allan:Janssen Ortho: Research Funding.

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Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression

Blood ◽

10.1182/blood.v118.21.1831.1831 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1831-1831 ◽

Cited By ~ 1

Author(s):

Michael Mangone ◽

Luigi Scotto ◽

Enrica Marchi ◽

Owen A. O'Connor ◽

Hearn J. Cho

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Research Funding ◽

Folate Metabolism ◽

Nog Mice ◽

Induced Apoptosis ◽

Dose Dependent ◽

Functional Biomarkers

Abstract Abstract 1831 Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4–7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM. Disclosures: O'Connor: Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

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Aurora Kinases in Childhood Acute Leukemia: The Promise of Aurora Kinase B As Drugable Target

Blood ◽

10.1182/blood.v118.21.1476.1476 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1476-1476

Author(s):

Stefanie A. Segers ◽

C. Michel Zwaan ◽

Carla Exalto ◽

Mirjam W.J. Luijendijk ◽

Valerie S. Calvert ◽

...

Keyword(s):

Acute Leukemia ◽

Cell Lines ◽

Aurora Kinase ◽

Selective Inhibitor ◽

Leukemic Cells ◽

Aurora Kinases ◽

Aurora Kinase B ◽

Pediatric All ◽

Childhood Acute Leukemia

Abstract Abstract 1476 AIM: Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Materials & Methods: Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay. Results: AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p<0.0002), especially in ALL cases with an E2A-PBX1 translocation (p<0.0001) than in nBM mononuclear cells. Silencing of AURKA by shRNA and by LNA-oligonucleotide caused no or only minor growth delay in several cell lines reflecting genetic subtypes typically found in pediatric ALL and AML. In contrast, silencing of AURKB resulted in proliferation arrest and apoptosis in these cells. Furthermore, 18 out of 20 ALL and AML cell lines tested were highly sensitive to the AURKB-selective inhibitor Barasertib-hQPA in the nanomolar range (IC50 = 19–233 nM) whereas less sensitivity was seen for other inhibitors. Conclusion: These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML. Disclosures: No relevant conflicts of interest to declare.

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Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽

10.1182/blood.v122.21.275.275 ◽

2013 ◽

Vol 122 (21) ◽

pp. 275-275

Author(s):

Siobhan Glavey ◽

Salomon Manier ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Yuji Mishima ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Myeloma Cells ◽

Sialyl Lewis ◽

And Migration ◽

Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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