The targeting of the immune system through immunotherapies to prevent tumor tolerance and immune suppression are at the front lines of breast cancer treatment and research. Human and laboratory ...studies have attributed breast cancer progression and metastasis to secondary organs such as the bone, to a number of factors, including elevated levels of prostaglandin E2 (PGE2) and the enzyme responsible for its production, cyclooxygenase 2 (COX2). Due to the strong connection of COX2 with immune function, we focused on understanding how variance in COX2 expression manipulates the immune profile in a syngeneic, and immune-competent, mouse model of breast cancer. Though there have been correlative findings linking elevated levels of COX2 and Tregs in other cancer models, we sought to elucidate the mechanisms by which these immuno-suppressive cells are recruited to breast tumor and the means by which they promote tumor tolerance.
To elucidate the mechanisms by which exacerbated COX2 expression potentiates metastasis we genetically manipulated non-metastatic mammary tumor cells (TM40D) to over-express COX2 (TM40D-COX2). Over-expression of COX2 in this mouse breast cancer model resulted in an increase in bone metastasis (an observation that was ablated following suppression of COX2 expression) in addition to an exacerbated Treg recruitment in the primary tumor. Interestingly, other immune-suppressive leukocytes, such as myeloid derived suppressor cells, were not altered in the primary tumor or the circulation. Elevated levels of PGE2 by tumor cells can directly recruit CD4+CD25+ cells through interactions with their EP2 and/or EP4 receptors, an effect that was blocked using anti-PGE2 antibody. Furthermore, increased Treg recruitment to the primary tumor contributed to the greater levels of apoptotic CD8+ T cells in the TM40D-COX2 tumors.
Due to the systemic effects of COX2 inhibitors, we propose targeting specific EP receptors as therapeutic interventions to breast cancer progression.
Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of ...individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-β. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.
Multiple clinical risk prediction tools for hospital acquired venous thromboembolism (HA-VTE) have been developed. The objectives of this study were to develop and assess the feasibility of data ...extraction from Electronic Medical Records (EMR) from an enterprise database warehouse (EDW) and to test the validity of a previously developed Pediatric Clot Decision Rule (PCDR). This single-center prospective observational cohort study was conducted between March 2016 and March 2017 and included eligible patients admitted to the intensive care units. Risk score was calculated using the PCDR tool. Sensitivity, specificity, positive and negative predicted value (PPV and NPV) were calculated based on a cut‐point of 3. A total of 2822 children were eligible for analysis and 5.1% (95% CI 4.2–6.2) children had a PCDR score of 3. Children with PCDR score of ≥ 3 had a 3 times higher odd of developing VTE compared to those with scores < 3 (OR 3.1; 95% CI 1.93–4.80; p < 0.001). The model performance showed that at the cutoff point of ≥ 3, both the specificity and sensitivity of the PCDR in predicting VTE was 69% and NPV of 98%. We successfully demonstrated using our EDW to populate a research database using an automatic data import. A PCDR score of ≥ 3 was associated with VTE. Collaboration through large registries will be useful in informing practices and guidelines for rare disorders such as pediatric VTE.
Acute myeloid leukemia (AML) patients with NPM1 mutations demonstrate a superior response to standard chemotherapy treatment. Our previous work has shown that these favorable outcomes are linked to ...the cytoplasmic relocalization and inactivation of FOXM1 driven by mutated NPM1. Here, we went on to confirm the important role of FOXM1 in increased chemoresistance in AML. A multiinstitution retrospective study was conducted to link FOXM1 expression to clinical outcomes in AML. We establish nuclear FOXM1 as an independent clinical predictor of chemotherapeutic resistance in intermediate-risk AML in a multivariate analysis incorporating standard clinicopathologic risk factors. Using colony assays, we show a dramatic decrease in colony size and numbers in AML cell lines with knockdown of FOXM1, suggesting an important role for FOXM1 in the clonogenic activity of AML cells. In order to further prove a potential role for FOXM1 in AML chemoresistance, we induced an FLT3-ITD-driven myeloid neoplasm in a FOXM1-overexpressing transgenic mouse model and demonstrated significantly higher residual disease after standard chemotherapy. This suggests that constitutive overexpression of FOXM1 in this model induces chemoresistance. Finally, we performed proof-of-principle experiments using a currently approved proteasome inhibitor, ixazomib, to target FOXM1 and demonstrated a therapeutic response in AML patient samples and animal models of AML that correlates with the suppression of FOXM1 and its transcriptional targets. Addition of low doses of ixazomib increases sensitization of AML cells to chemotherapy backbone drugs cytarabine and the hypomethylator 5-azacitidine. Our results underscore the importance of FOXM1 in AML progression and treatment, and they suggest that targeting it may have therapeutic benefit in combination with standard AML therapies.
Background: Venous thromboembolism (VTE) in critically ill children is associated with increased morbidity and mortality. Therefore, significant efforts to identify children with a predisposition to ...VTE are being made so that prevention strategies can be implemented. To identify these children a priori, our group developed the Peds Clot Clinical Decision Rule based on exposure to known VTE risk factors (Sharathkumar et al Journal of Thrombosis and Haemostasis, 10: 1326-1334). Since this decision rule was developed using a retrospective case-control study design, we performed prospective validation of the PCDR utilizing the automatic data-collection tool available via our institution's Computer Data Warehouse (CDW). The interim analysis(Prospective Validation of the Peds Clot Clinical Decision Rule Pdcr in Hospital-Acquired Venous Thromboembolism: An Interim Analysis Blood 2016 128:3812) showed that three of the six variables, immobility, blood stream infection (BSI), and oral contraceptive pill (OCP) use, required manual entry. To capture immobility objectively, we took advantage of a recent initiative to record immobility of patients 13 and older at Lurie Children's Hospital. This report summarizes the analysis of this ongoing validation effort.
Objective: 1. To validate the Peds Clot Clinical Decision Rule PCDR utilizing automatic data collection at Lurie Children's hospital; 2. To evaluate whether the sensitivity and specificity of PDCR improves after objective assessment of immobility.
Methods: The Computer data warehouse (CDW) facility was used to directly import relevant variables into the database for our study period of 03/01/16-03/31/17. Inclusion criteria for the study were Intensive Care Unit (Pediatric, Cardiac, Neonatal) stay for at least 48 hours or more. The following demographic and clinical variables were included in the dataset: age, sex, ethnicity, date of admission and discharge, central venous catheter (CVC), BSI, immobility (defined as being incapable of ambulating without assistance), OCP use, mechanical ventilation, and length of hospitalization (LOH). Risk factors were scored as per PCDR rule1. Chi-square tests were used to examine the association between the potential risk factors and VTE. Risk factors with an association to VTE in univariate analysis were included in multivariate logistic regression model. PCDR model performance was evaluated by reporting the sensitivity and specificity. Additional independent multivariate analysis was performed to explore the role of other variables in the development of VTE.
Results: A total of 2836 children were eligible for analysis. The demographic and clinical features are detailed in Table 1. Of 2836 patients, 141 (4.9%) were identified as having VTE and 881 (31%) had a CVC. LOH, presence of CVC, and BSI continued to be significantly associated with a VTE event. There was an association between VTE and immobility, but it did not reach statistical significance. The model performance showed that at the cutoff point of 3, the specificity and sensitivity of the PDCR in predicting VTE is 62% and 70% respectively. This is an improvement from our interim analysis report which had a specificity of 75% but a very low sensitivity (37%). Presence of a CVC was independently associated with VTE risk (AOR=4.33; 95% CI=2.88, 6.52). Caucasian race emerged to be associated with VTE and but did not retain its significance in multivariate analysis. Interestingly, other variables (immobility, length of stay, OCP) did not show an association with VTE in the multivariable analysis.
Conclusions: Our study confirms that automatic data collection is feasible with electronic medical records and data-ware house facility. Diligent efforts to collect standardized data for immobility resulted in only a minor improvement in the performance of the PCDR. This suggests that unlike in the general hospital population, underlying disease processes may be more important determinants of a procoagulant phenotype in critically ill patients. We plan to assess these processes in our next analysis. It is noteworthy that prospective data-collection improved the sensitivity of PCDR. This will help clinicians closely monitor this population and reduce or avoid exposure to risk factors for VTE.
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Sharathkumar:Shire: Consultancy; CSL Behring: Consultancy.
Background: Venous thromboembolism (VTE) is an emerging hospital acquired complication in pediatric hospitals. Substantial efforts have been made to identify high-risk population based on exposure to ...risk-factors for VTE. Our group developed the PedsClot Clinical Decision Rule (Sharathkumar et al Journal of Thrombosis and Haemostasis, 10: 1326-1334) to identify this high-risk population based on a case-control study but this has not been validated yet.
Objective: To validate thePedsClot Clinical Decision Rule PDCR through prospective collection of data in a tertiary pediatric hospital. This study reports our findings of interim analyses for process evaluation.
Methods: This prospective data collection was performed using the Lurie Children’s hospital automatic data import facility through the Enterprise Data Warehousestudy period: 02/01/2012-12/31/14. Real time data was added to a research database of consecutive admissions based on following inclusion criteria: > 48 hour stay in the Intensive Care (Pediatric, Cardiac, Neonatal), Hematology Oncology and Infectious Disease units. Following variables were included in the dataset: age, sex, ethnicity, date of admission and discharge, ICU admission, central venous catheter (CVC), blood stream infection, immobilization, oral contraceptives, mechanical ventilation> 12hrs, and length of stay. Risk factors were weighed and scored as per PDCR rule. Chi-square tests were used to examine the association between the potential risk factors and VTE. PDCR model performance was evaluated by reporting the sensitivity and specificity.
Results: A total of 1722 children were eligible for the interim analyses. The demographic and clinical features of the dataset analyses and the odds ratio OR are detailed in Table 1 and 2 respectively. Of 1722 patients, 57 (3.3%) were identified as VTE, 50% were admitted to the ICU and 51% had a CVC. VTE was associated with age ≥ 13 years (AOR=2.3; 95% CI=1.3, 4.0), ICU admission (AOR=2.4; 95% CI=1.3, 4.4), and CVC (AOR=1.9; 95% CI=1.1, 3.3). The model performance showed that at the cut off point of 3, the specificity of the PDCR in predicting VTE was 75% but had a low sensitivity (37%). Risk prediction variables such as assessment of immobilization, use of oral contraceptives and prediction of hospital stay required manual entry into the datasheet; this makes data-capture labor intensive, especially for larger datasets.
Conclusions: This study utilized technological advances and database warehouse facility to validate PDCR. This interim data analyses indicates that in real life, PDCR has a high specificity but poor sensitivity in identifying children with predisposition for VTE. We are currently working to refine the risk model which may lead to a better model performance.
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Sharathkumar:Bayer, Baxter, CSL Behring: Consultancy.
Down syndrome-associated acute megakaryoblastic leukemia (DS-AMKL) affects 1 in 500 children with DS and associates uniformly with somatic mutations in GATA1. DS-AMKL is often treated successfully; ...however, treatments can cause long-term side effects, such that mechanistic insights and targeted therapies are needed.
Recent studies report a recurring L340F mutation in DCAF7 in DS-AMKL, at the C-terminus of the protein. A highly conserved scaffold protein, DCAF7 is a cofactor for the chromosome 21-encoded kinase, DYRK1A, and shares 100% protein sequence homology among vertebrates. In our efforts to characterize the normal function of DCAF7 in hematopoiesis and its contribution to AMKL, we have identified an unreported PDZ-binding motif (PBM) in DCAF7, which is perturbed by the L340F mutation. Overexpression of mutant (DCAF7mut), but not wild type DCAF7 (DCAF7wt), in a mouse model of DS with a Gata1 mutation leads to rapid onset of dysplastic disease characterized by peripheral blood leukopenia and thrombocytopenia, with normal red cell numbers. Bone marrow of affected mice harbors abnormal, hyperproliferative erythroid progenitors and diminished, hyperchromatic megakaryocytes, with stark depletion of granulocytic and lymphoid lineages and widespread edema. Splenic architecture is grossly abnormal, with a complete absence of white pulp, proliferation of erythroid precursors, and significant numbers of apoptotic, hyperchromatic megakaryocytes.
Conversely, targeted deletion of Dcaf7 in hematopoiesis shifts the myeloid-erythroid balance toward the myeloid lineage, with increased granulopoiesis and hyperchromatic megakaryocytes in the bone marrow and lower white blood cell numbers and dramatic thrombocytopenia in the peripheral blood. Splenic architecture is largely normal, although flow cytometry reveals defective B- and T- cell development.
The bone marrow knockout phenotype of Dcaf7 is strongly reminiscent of the cellular phase of primary myelofibrosis (PMF). Combined with bone marrow overexpression phenotypes, which resemble hemophagocytic syndrome, our data suggest that DCAF7 is critically important for maintaining a healthy bone marrow environment and regulating blood cell lineage choice. In order to identify functional binding partners of the DCAF7 PDZ-binding motif (PBM) that may support these functions, we performed IP mass spectrometry using DCAF7wt or DCAF7mut as bait and found two highly conserved PDZ-domain containing scaffold proteins, PAR3 and PDLIM5, that interact robustly with DCAF7wt, but not with DCAF7mut. While both DCAF7wt and DCAF7mut are able to associate with DYRK1A, the ability of DCAF7 to bring DYRK1A activity to the PAR3 and PDLIM5 signaling hubs affects the composition of DCAF7-scaffolded transcriptional complexes and downstream transcriptional outcomes.
Through RNA-sequencing and Ingenuity Pathways Analysis, overexpression of both DCAF7wt and DCAF7mut in mouse bone marrow leads to the induction of interferon response genes; however, DCAF7wt also induces a pro-apoptotic gene program, whereas DCAF7mut instead promotes survival gene expression and developmental reprogramming. Consistently, expression of DCAF7wt in bone marrow progenitors causes rapid expansion and increased initial colony forming ability, but decreased replating potential compared with vector control. Conversely, expression of DCAF7mut causes increased proliferation and colony formation accompanied by enhanced replating, indicating greater self-renewal conferred by DCAF7mu.
Our data indicate DCAF7 regulates the balance of proliferation, differentiation and apoptosis in hematopoietic cells and that the PBD of DCAF7 is critically important for these functions, connecting DCAF7 to signaling hubs that translate environmental cues into appropriate transcriptional outcomes. Furthermore, DCAF7 disruption causes hematopoietic disease reminiscent of human pathology, suggesting it may have broad significance in hematopoietic malignancy. Finally, our data support a model wherein DCAF7 mutation facilitates the development of malignancy by promoting the survival and proliferation of mutant clones in the bone marrow and fundamentally altering the bone marrow microenvironment.
No relevant conflicts of interest to declare.
The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling ...pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.
Objectives:
Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces genes critical in executing the mitotic program. The FOXM1 regulatory network is a major predictor of ...adverse survival outcomes across 18000 human tumor expression signatures (Gentles. Nat Med 2015). FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines the cellular localization of FOXM1. In the OCI-AML3 cell line and in AML primary samples with mutant alleles of NPM1, we have shown that FOXM1 and mutant NPM are re-localized to the cytoplasm (Khan. Leukemia 2017). We postulated that nuclear export of FOXM1 may underlie the chemotherapy responsiveness in NPM1mut patients. We conducted a multi-institution study to investigate the nuclear expression level of FOXM1 as a predictor of chemotherapy resistance in AML and validate it as a therapeutic target.
Methods:
Adult patients diagnosed with intermediate-risk AML were identified at Northwestern Memorial and the University of Illinois Hospitals. A total of 102 patients were included in the study with equivalent representation from each institution. Clinical data were collected by chart review, and corresponding bone marrow biopsy samples were retrieved under an IRB approved protocol. Tissue sections were stained with a FOXM1 antibody and scanned images were analyzed using HALO 2.0 software. All biopsy specimens were reviewed by a hematopathologist for adequacy. The percentage of total nuclei expressing FOXM1, nuclear FOXM1 intensity (optical density), and the FOXM1 nuclear:cytoplasmic ratio were quantified. Statistical analysis was performed by the Design and Analysis Core, UIC.
Results:
Patient characteristics are summarized in Table 1. Out of 102 patients, 77 (75%) achieved a complete remission with or without count recovery (CR/CRi). We used lines of induction therapy needed to achieve remission as a measure of chemotherapy resistance. Patients were stratified as needing 1 or >1 line of induction therapy. Induction therapy included cytarabine and anthracycline in 82 cases and hypomethylator in 19 cases.
We found that patients needing >1 line of induction therapy had substantially higher levels of nuclear FOXM1 in their diagnostic bone marrow biopsy compared to patients who responded to first-line therapy (20.4% vs. 11.4% positive nuclei; p= 0.066). In logistic regression analysis, the percentage of FOXM1 positive nuclei (OR 1.85 for 10% increase in positive nuclei, p=0.02) and average nuclear intensity (OR 2.18 for 0.1 U increase in OD, p=0.05) were able to predict resistance to first-line chemotherapy. Due to variation in post-induction therapies, the institutions were considered separately for survival analysis. Nuc:Cyto FOXM1 ratio was able to predict inferior overall survival in a single institution cohort analyzed (n=45) in a model incorporating standard clinicopathologic prognostic factors. Multivariate regression analysis is shown in Table 2.
We then proceeded to validate nuclear FOXM1 as a therapeutic target in AML. Short hairpin knockdown of FOXM1 in AML cell lines results in decreased colony size and number (KG-1 shFOXM1: 0.35 normalized to control, p=0.01; MV4-11 shFOXM1: 0.61, p=0.03). We have shown that proteasome inhibitors stabilize HSP70 which is a negative regulator of FOXM1 (Halasi. JBC 2016). The novel proteasome inhibitor ixazomib was tested for in vitro effect on FOXM1 in AML cells. Ixazomib inhibits the transcriptional activity of FOXM1 using a luciferase reporter cell line with inducible FOXM1. Treatment with ixazomib significantly decreases FOXM1 mRNA and inhibits FOXM1 protein expression in AML cell lines and patient samples (n=12) resulting in increased apoptosis by caspase cleavage. This is consistent with the FOXM1 auto-regulation loop (Halasi. Cell Cycle 2009). The therapeutic efficacy of ixazomib in AML was then tested in vivo. Single agent treatment with ixazomib compared to vehicle in an murine xenograft model of AML showed significant reduction in tumor volume after 3 weeks of treatment (0.89cm3 vs 1.62cm3, n=8 per group) and downregulation of FOXM1 expression.
Conclusion:
Our work shows for the first time that nuclear FOXM1 confers chemotherapy resistance in a clinically homogenous group of intermediate risk AML patients. Moreover we show that ixazomib, a well-tolerated novel proteasome inhibitors has therapeutic efficacy in AML models that correlates with suppression of FOXM1.
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Khan:Teva: Speakers Bureau; Takeda: Research Funding; Gilead: Consultancy.
Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of ...patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF.
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