Brigham L. Bahr

Different expression of placental pyruvate kinase in normal, preeclamptic and intrauterine growth restriction pregnancies

INTRODUCTION Preeclampsia (PE) and intrauterine growth restriction (IUGR) are two diseases that affect pregnant women and their unborn children. These diseases cause low birth weight, pre-term delivery, and neurological and cardiovascular disorders in babies. Combined they account for 20% of preterm deliveries. Pyruvate kinase M2 (PKM2) is a metabolism enzyme found in developing embryonic and cancer tissues. Our objective is to determine the expression of PKM2 in human PE and IUGR compared to normal pregnancies. Understanding expression of PKM2 in PE and IUGR could help us to better understand the mechanisms and find treatments for PE and IUGR. METHODS Human placental tissues were obtained for PKM2 determination and analyzed by immunohistochemistry, Western blot, and a pyruvate assay. Placental samples were homogenized and cytoplasmic and nuclear proteins were extracted for Western blot analysis. RESULTS Preeclampsia samples had elevated levels of p-PKM2, p-ERK, and ERK in the cytoplasm. Beta-catenin and lactose dehydrogenase (LDH) were also elevated in preeclampsia placenta samples. DISCUSSION AND CONCLUSION We conclude that PKM2 is expressed in normal, PE and IUGR pregnancies. Also, that this expression is increased in the PE placenta at delivery. These results suggest placental metabolism through PKM2 could play a role in human preeclampsia.

Abstract 2698: Combination strategies to target super enhancer transcriptional activity by CDK9 and BRD4 inhibition in acute myeloid leukemia

The super enhancer complex (SEC) is a group of transcription regulatory proteins that coordinate the expression of genetic programs which determine cell identity and drive disease states, such as cancer. In acute myeloid leukemia (AML), SECs have been shown to turn on transcriptional programs that drive tumorigenesis and disease progression. The SEC is replete with potential therapeutic targets that have been the focus of many drug development efforts; including cyclin-dependent kinases (CDK), bromodomain proteins (BRD), histone deacetylases (HDAC), and histone methyltransferases (HMT). SEC-regulated transcription begins as CDK9/cyclin T1 is recruited from an inhibitory complex by BRD4 and brought to the transcriptional start site of genes. CDK9 phosphorylates RNA polymerase II, releasing it from the SEC and leading to transcriptional elongation and gene expression. Considering the close association of CDK9 and BRD4, we hypothesized that the combination of CDK9 and BRD4 inhibitors would have synergistic effects, particularly in AML, a disease largely driven by SEC function. Alvocidib is a potent CDK9 inhibitor with validated clinical activity in AML from multiple Phase II studies in over 400 patients. Additionally, BRD4 inhibitors have demonstrated early promise in clinical studies with a focus on AML. We found that CDK9 inhibitors combined with bromodomain inhibitors produced a synergistic effect by inhibiting the SEC more effectively than either of these compounds alone. For example, cell viability studies with various combinations resulted in an increase in potency. This was observed with alvocidib combined with JQ-1 (BRD4 inhibitor) in MV4-11 AML cells. Furthermore, the combination of alvocidib with JQ-1 completely abrogated SEC function, as measured by c-myc expression through RT-qPCR. Similar results were achieved with other combinations of CDK9 and BRD4 inhibitors. The alvocidib and JQ-1 combination was also evaluated in an MV4-11 mouse xenograft model. As single agents, alvocidib (2.5 mg/kg) exhibited a 44% tumor growth inhibition and JQ-1 (25 mg/kg) a 1% growth inhibition. When these two doses were combined there was 100% tumor growth inhibition. These data, primarily focused on alvocidib and JQ-1, suggest a strong rational for combining CDK9 and BRD4 inhibitors as a treatment strategy for AML. Furthermore, these findings could be more broadly applied to additional therapeutic targets in the SEC, such as DOT1L and HDACs. These strategies yield synergistic effects at inhibiting SEC function and are highly active in tumor growth studies of AML in vivo. Clinical studies utilizing these combination strategies are the next steps to further explore this approach. Citation Format: Brigham L. Bahr, Kyle S. Maughan, Katherine K. Soh, Jeremiah J. Bearss, Wontak Kim, Peter Peterson, Clifford Whatcott, Adam Siddiqui-Jain, Steve L. Warner, David J. Bearss. Combination strategies to target super enhancer transcriptional activity by CDK9 and BRD4 inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2698. doi:10.1158/1538-7445.AM2015-2698

Abstract 3604: Targeting the PIM kinases in combination with BTK inhibition is synergistic in preclinical models of B-cell malignancies

BTK inhibitors (e.g. ibrutinib) have significantly impacted the treatment of B-cell malignancies in a positive way. Single agent response rates with ibrutinib are 65% or higher in B-cell lymphomas and chronic lymphocytic leukaemia with the majority of patients enjoying a prolonged duration of response. Continued clinical development is needed, however, as most patients achieve only a partial response from their treatment and ultimately patients become refractory to ibrutinib leading to relapse and disease progression. Targeted combinations with ibrutinib could potentially increase the number of patients undergoing complete remission and combat emergent resistant mechanisms. The PIM family (1, 2, and 3) are serine/threonine kinases that have proven to be oncogenic in-part due to their ability to suppress c-Myc induced apoptosis. The PIM kinases have emerged as important regulators of drug resistance in multiple cancer types. Tolero Pharmaceutical9s second generation PIM Kinase inhibitor, TP-3654 has exhibited favorable activity in preclinical models of prostate cancer, AML, and lymphoma. Due to the signaling crosstalk between BTK and PIM through the STAT transcription factors, we hypothesized that synergies may arise through the simultaneous targeting of both kinases. Here, we report a significant increase in drug activity when a BTK inhibitor (ibrutinib) was combined with TP-3654 in various lymphoma cell lines. In Granta-519 cells, the IC50 of ibrutinib decreased 3.5-fold, from 0.7 μM to 0.2 μM, when cultured in combination with a subtoxic concentration of TP-3654 (300 nM). Similarly, the IC50 of TP-3654 decreased 6-fold, from 2.4 μM to 0.4 μM, when cells were cultured in combination with a subtoxic concentration of ibrutinib (100 nM). BTK is known to attenuate the activity of the transcription factor STAT3, a major regulator of PIM kinase levels in cells. Due to this, mechanistic studies focused on analyzing the STAT3 pathway are ongoing to determine the downstream effects of using ibrutinib and TP-3654 in combination. Several lymphoma xenograft studies are also ongoing to further explore this combination in vivo. These results provide a strong rationale that inhibitors of PIM and BTK could be used in combination for the treatment of B-cell malignancies and other B-cell mediated diseases. Citation Format: Jeremiah J. Bearss, Brigham L. Bahr, Katie K. Soh, Peter W. Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. Targeting the PIM kinases in combination with BTK inhibition is synergistic in preclinical models of B-cell malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3604. doi:10.1158/1538-7445.AM2015-3604

Inhibition of AXL kinase reverses the mesenchymal phenotype in leukemia cells through the disruption of retinoic acid signaling

Chronic lymphocytic leukemia: Risk factors and complications. Journal of Tumor Research.Vol.6 No.1: 1. Background & Definition: Chronic lymphocytic leukemia (CLL) results from an acquired (not present at birth mutation (change)) to the DNA of a single marrow cell that develops into a lymphocyte. Scientists do not yet understand what causes this change. Once the marrow cell undergoes the leukemic change, it multiplies into many cells. CLL cells grow and survive better than normal cells; over time, they crowd out normal cells. The result is the uncontrolled growth of CLL cells in the marrow, leading to an increase in the number of CLL cells in the blood. The leukemic cells that accumulate in the marrow in people with CLL do not prevent normal blood cell production as extensively as is the case with acute lymphoblastic leukemia. Risk Factors: First-degree relatives of patients with CLL are three to four times more likely to develop CLL than people who do not have first-degree relatives with the disease. Old age is second risk factor of CLL development. Sign and symptoms: Early, some people with CLL do not have any symptoms. The disease may be suspected because of abnormal results from blood tests that were ordered either as part of an annual physical or a medical examination for an unrelated condition. An unexplained elevated white blood cell (lymphocyte) count is the most common finding that leads a doctor to consider a CLL diagnosis. Diagnosis: The diagnosis of CLL is usually evident from the results of blood cell counts and an examination of blood cells. A bone marrow aspiration and biopsy generally are not needed to make a diagnosis of CLL provided the red blood cells and platelets are normal. “Immunopheno typing” (or flow cytometry) of lymphocytes is an important process used to diagnose CLL, and other types of leukemia and lymphoma, by comparing the cancer cells to normal immune cells. Staging for CLL helps doctors to both assess how the disease is expected to progress over time and also to develop a treatment plan. Complications: CLL or CLL Treatment: Infections are a common complication for people with CLL. Anemia (low numbers of red blood cells) is a common side effect of chemotherapy. In about 3 to 5 percent of people with CLL, the disease transforms into an aggressive lymphoma (Richter Transformation) because of a change in the characteristics of the CLL cells. About 15 percent of people with CLL develop prolymphocytic leukemia. Some people with CLL produce a type of antibody that works against their own cells (Autoimmune HemolyticAnemia). People with CLL have a higher risk than the general population of developing a second cancer. Conclusion: People with CLL need regular medical follow- up after they have completed treatment. It is important to assess the full effect of therapy as well as to identify any return of progressive disease that may require additional therapy.Ceaseless lymphocytic leukemia (CLL) is the most well-known leukemia in grown-ups. It’s a kind of disease that begins in cells that become certain white platelets (called lymphocytes) in the bone marrow. The malignancy (leukemia) cells start in the bone marrow yet then go into the blood. In interminable leukemia, the cells can develop halfway (and more resemble ordinary white platelets). be that as it may, not totally. These cells may look genuinely typical, however they’re definitely not. They by and large don’t battle contamination just as expected white platelets do. The leukemia cells endure longer than ordinary cells, and develop, jamming out typical cells in the bone marrow. It can require some investment before incessant leukemiascause issues, and a great many people can live with them for a long time. However, interminable leukemias will in general be harder to fix than intense leukemias. The regular type of CLL begins in B lymphocytes. However, there are some uncommon sorts of leukemia that share a few highlights with CLL. Prolymphocytic leukemia (PLL): In this sort of leukemia the disease cells are a great deal like ordinary cells called prolymphocytes. These are juvenile types of B lymphocytes (B-PLL) or T lymphocytes (T-PLL). Both B-PLL and T-PLL will in general develop and spread quicker than the standard kind of CLL. A great many people with it will react to some type of treatment, however after some time they will in general backslide (the disease returns). PLL may create in somebody who as of now has CLL (in which case it will in general be more forceful), yet it can likewise happen in individuals who have never had CLL. Enormous granular lymphocyte (LGL) leukemia: This is another uncommon type of constant leukemia. The disease cells are enormous and have highlights of either T lymphocytes or another sort of lymphocyte called characteristic executioner (NK) cells. Most LGL leukemias are moderate developing, yet a modest number are more forceful (they develop and spread rapidly). Medications that smother the resistant framework may help, however the forceful sorts are extremely difficult to treat. Furry cell leukemia (HCL): This is uncommon malignant growth of the lymphocytes that will in general advancement gradually. The malignancy cells are a kind of B lymphocyte however they’re not quite the same as those seen in CLL. There are likewise significant contrasts in side effects and treatment. This sort of leukemia gets its name from the manner in which the cells look under the magnifying instrument - they have fine projections on their surface that make them look “bristly.”Objective: Liver cancer is the fifth most common cancer in men and the seventh in women. During the past 20 years, the incidence of HCC has tripled while the 5-year survival rate has remained below 12%. The presence of circulating tumour cells (CTCs) reflects the aggressive nature of the tumour during the development of the HCC. CTCs detection and identification can be used to estimate prognosis and may serve as an early marker to assess antitumor activity of treatment. CTCs are an interesting source of biological information in order to understand dissemination, drug resistance, and treatment-induced cell death. The aim is to estimate the CTCs (AFP mRNA & TGFβ1 mRNA) in the peripheral blood of patients with HCC as an early non invasive marker of HCC detection and prognosis. Patients and methods: The study was done on 100 patients, 58 patients with hepatocellular carcinoma (HCC) and 42 patients with liver cirrhosis (LC), and 20 healthy volunteers as a control group. Detailed clinical history and examination were carried out. Complete blood count, liver function test, serum Albumin, serum AFP, AFP mRNA, serum TGF-β1 and TGF-β1 mRNA were measured.Abdominal ultrasound was done for all studied subjects and CTscan Abdomen for those with HCC to determine the size and number of tumour. Results: The detection rate of AFP mRNA was 39.7%, 11.9% and5% in patients with HCC, LC and control subjects respectively with a significant expression in HCC patients compared to other groups. Also TGF-β1 mRNA expression was significantly high in HCC cases with detection rate 60.3%, 14.3% in HCC and LC respectively while it was not detected in the controls. Both CTC were correlated with Milan criteria.The serum levels of AFP and TGFβ1 was significantly higher in HCC patients. Conclusion: TGF-β1 mRNA is a more reliable marker for diagnosis of HCC and if combined with AFP mRNA yielded better prediction of HCC prognosis. Since HCC is among the cancers with worst prognosis, early diagnosis and treatment are essential for better outcome. [Nashwa Sheble, Gehan Hamdy, Moones A Obada, Gamal Y Abouria, Fatma Khalaf, Enas A Khattab. The Value of Measurement of Circulating Tumor Cells in Hepatocellular Carcinoma. Life Sci J 2013;10(4):1-11] (ISSN: 1097-8135). http://www.lifesciencesite.com. 1Hemophagocyticlymphohistiocytosis (HLH) is a rapidly progressive, life-threatening syndrome of excessive immune activation. Prompt initiation of treatment for HLH is essential for the survival of affected patients. Although haematologist oncologists will treat these patients, but general paediatricians, immunologists, gastroenterologists, infectious men and even internists and adult oncologists should be familiar with diagnosis of this disease which has high morbidity and mortality; and its treatment before progression of neurologic complications is helpful. Presentation of HLH will be mentioned to be considered in the differential diagnosis of critically ill patients with prolonged fever, splenomegaly, cytopenia, jaundice, coagulopathy, etc. Method: English language large data bases including PubMed Central (Medline), EMBASE, Science Direct, ISI, ISC and Google Scholar were reviewed using these keywords: HemophagocyticLymphohistiocytosis, Diagnosis and/or treatment of HLH, genetic predisposition to HLH, Children or Adults with HLH. Fifty seven review articles were enrolled. Results: OVERVIEW AND INDICATIONS FOR TREATMENT: Hemophagocytic lymphohistiocytosis (HLH) is a dynamic disorder of unchecked insusceptible initiation and tissue harm. In the event that left untreated, patients with HLH make due for just a couple of months, because of dynamic multi-organ disappointment. Regularly, the best boundary to treatment and a fruitful result for people with HLH is a deferral in finding. A few parts of the clinical presentation of HLH add to this postponement, including the uncommonness of the disorder, the variable clinical presentation, and the absence of specificity of the clinical and research facility discoveries. Diagnostic criteria for HLH include molecular testing consistent with HLH or 5 of 8 of the following criteria: fever, splenomegaly, cytopenias affecting ≥2 lineages, hyperferritinemia, hypertriglyceridemia and/ or hypofibrinogenemia, hemophagocytosis (in bone marrow, spleen, or lymph node), impaired NK cell function, and elevated soluble CD25 (sCD25) (ie, sIL2R). Additional discoveries that are regular are transaminases, coagulopathy, hypernatremia, edema, rash, hypoalbuminemia, hoisted lactate dehydrogenase (LDH), C-responsive protein, and d-dimer, expanded lowthickness lipoprotein, diminished high-thickness lipoprotein, lifted cerebrospinal liquid protein and cells, and neurologic indications running from central shortages to adjusted mental status. Any patient with suspected HLH ought to be seen by a haematologist, and the individuals who are intensely sick ought to be exchanged eminently to an office where they can get HLH treatment.Hemophagocytic lymphohistiocytosis (HLH), otherwise called haemophagocytic lymphohistiocytosis, and hemophagocytic or haemophagocytic syndrome, is an exceptional hematologic issue seen more regularly in kids than in grown-ups. It is a perilous sickness of extreme hyperinflammation brought about by uncontrolled multiplication of actuated lymphocytes and macrophages, portrayed by expansion of morphologically kindhearted lymphocytes and macrophages that discharge high measures of provocative cytokines. It is delegated one of the cytokine storm conditions. There are acquired and non-acquired (procured) reasons for hemophagocytic lymphohistiocytosis (HLH).The beginning of HLH happens younger than one year in roughly 70 percent of cases. Familial HLH ought to be suspected if kin are determined to have HLH or if side effects repeat when treatment has been halted. Each full kin of a youngster with familial HLH has a twenty-five–percent possibility of building up the sickness, a 50% possibility of conveying the deficient quality (which is once in a while connected with any danger of malady), and a twenty-five–percent possibility of not being influenced and not conveying the quality imperfection. Patients with HLH, particularly when untreated, may require escalated treatment. In this manner, HLH ought to be remembered for the differential conclusion of emergency unit with cytopenia and hyperferritinemia. Patients in the previous phases of HLH are much of the time hospitalized at interior medication wards. HLH clinically shows with fever, augmentation of the liver and spleen, broadened lymph hubs, yellow staining of the skin and eyes, and a rash. Research center discoveries may incorporate raised triglyceride levels, low fibrinogen levels, transaminitis, and raised ferritin levels (among others). Essential HLH is brought about by loss of capacity, (for example inactivating) changes in qualities that code for proteins cytotoxic T cells and NK cells use to murder focused on cells, for example, those contaminated with microbes like the Epstein-Barr infection (EBV) or the Dengue virus. These transformations incorporate those in the accompanying qualities: UNC13D, STX11, RAB27A, STXBP2, LYST, PRF1 1, SH2D1A, BIRC4, ITK, CD27, and MAGT1. Auxiliary HLH (sHLH) is related with, and thought to be advanced, by threatening and non-harmful illnesses that in like manner debilitate the capacity of the safe framework capacity to assault EBV-tainted cells. Harmful issues related with optional HLH incorporate T-cell lymphoma, B-cell lymphoma, intense lymphocytic leukemia, intense myeloid leukemia, and myelodysplastic disorder. Non-harmful issues related with auxiliary HLH include: immune system issues, for example, adolescent idiopathic joint pain, adolescent Kawasaki ailment, fundamental lupus erythematosus, the adolescent beginning and grown-up beginning types of Still’s ailment, and rheumatoid arthritis; immunodeficiency issues, for example, extreme consolidated immunodeficiency, DiGeorge disorder, Wiskott–Aldrich condition, ataxia–telangiectasia, and dyskeratosis congenita); and contaminations brought about by EBV, cytomegalovirus, HIV/AIDS, microbes, protozoa, growths and potentially SARSCoV- 2. Secondary HLH may likewise result from iatrogenic causes, for example, bone marrow or other organ transplantations; chemotherapy; or treatment with immunosuppressing specialists About 33% of all HLH cases, ~75% of Asian HLH cases, and almost 100% of HLH cases brought about by transformations in SH2D1A (see X-connected lymphoproliferative ailment type 1) are related with, and thought activated or advanced by, EBV disease. These instances of HLH are delegated having a place with the class of Epstein–Barr infection related lymphoproliferative maladies and named EBV+ HLH.Beta-thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. Clinical features of beta-thalassemia include variably severe anemia and iron overload due to increased intestinal iron absorption, which may result in damage to vital organs. The hepatic peptide; hepcidin is a key regulator of iron metabolism in mammals. The present study aimed to determine the relationship between hepcidin expression and iron status in beta-thalassemia patients with hepatitis C virus infection. The study included 50 patients diagnosed as beta-thalassemia major (21 of them were HCV infected and 29 were HCV negative), in addition, 20 healthy subjects were enrolled in the study. The hepatic iron and hepcidin mRNA concentration in liver biopsy samples were measured, as well as serum ferritin, serum iron, hemoglobin and levels and serum hepcidin. Result showed remarkable decrease of serum and liver hepcidin mRNA expression in thalassemic patients as compared to controls, and showed a positive correlation with hemoglobin concentration, but negatively correlated with serum ferritin level and hepatic iron index (HII). In HCV infected patients, serum and liver hepcidin mRNA were markedly depressed in HCV positive beta-thalassemia cases, and positively correlated serum albumin and prothrombin concentrations, but inversely correlated with HII and fibrosis score. In HCV positive beta-thalassemia major patients, the hepcidin mRNA level was positively correlated with the synthetic function of the liver (namely serum albumin and prothrombin concentration) and with serum hepcidin level. While, both serum and hepcidin mRNA level was inversely correlated with HII and fibrosis score in these patients. These results suggest a possible role of hepcidin expression in iron overload in beta-thalassemia major, consequent disease progression and development of liver fibrosis.

Abstract 1875: A novel series of metabolic activators of PKM2 alter oncogene-meditated changes in tumor cell metabolism.

Rather than relying on oxidative phosphorylation for the generation of ATP, human tumor cells primarily utilize aerobic glycolysis to metabolize glucose (the Warburg effect). Pyruvate kinase is a metabolic enzyme that converts phosphoenolpyruvate (PEP) to pyruvate, catalyzing the rate-limiting step of glycolysis. The M1 isoform of pyruvate kinase (PKM1) that is the principal isoform in most adult differentiated tissues, while the PKM2 splice variant is the main isoform in embryonic tissues and in all cancer cells. Unlike the M1 splice form (PKM1), which is found in its tetrameric active form in cells, PKM2 is found in cells as an inactive dimer under normal physiological conditions. Tetramerization of PKM2 requires binding of the allosteric activator fructose-1,6-bisphosphate (FBP), an upstream glycolytic intermediate, resulting in a fully active enzyme. Regulation of PKM2 activity in cancer cells may allow glycolytic intermediates to be diverted into other biosynthetic pathways necessary for biomass production. PKM2 expression enhances tumorigenicity of cells while PKM1 expression represses it. This suggests that activators of PKM2 may have anti-tumor properties by forcing PKM2 to act more like PKM1. We have a series of small molecule PKM2 activators that exhibit low nM activation activity in biochemical and cell-based assays. These compounds increase pyruvate kinase activity in cancer cells and lead to an increase in pyruvate and ATP production. Our studies show that PKM2 activators inhibit the growth of lung cancer cell lines in vitro and in vivo and can reverse the metabolic changes induced by oncogenes such as k-Ras and c-Myc in lung cancer cells. The current lead compound was tested in established subcutaneously implanted A549 lung adenocarcinoma xenografts, where we observed a statistically significant 54% decrease in tumor growth, with no observable toxicity. These data suggest that this class of PKM2 activators is effective as tumor cell metabolic regulators with anti-tumor activity for lung cancer and potentially other malignancies. Citation Format: Brigham L. Bahr, Jenny Stevens, Spencer Squire, Christopher Moreno, Lee T. Call, Bret J. Stephens, Alexis Mollard, Steven L. Warner, David J. Bearss. A novel series of metabolic activators of PKM2 alter oncogene-meditated changes in tumor cell metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1875. doi:10.1158/1538-7445.AM2013-1875