Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common form of acute leukemia in children. Despite this, very little improvement in survival rates has been achieved over the past few decades. This is partially due to the heterogeneity of AML and the need for more targeted therapeutics than the traditional cytotoxic chemotherapies that have been a mainstay in therapy for the past 50 years. In the past 20 years, research has been diversifying the approach to treating AML by investigating molecular pathways uniquely relevant to AML cell proliferation and survival. Here we review the development of novel therapeutics in targeting apoptosis, receptor tyrosine kinase (RTK) signaling, hedgehog (HH) pathway, mitochondrial function, DNA repair, and c-Myc signaling. There has been an impressive effort into better understanding the diversity of AML cell characteristics and here we highlight important preclinical studies that have supported therapeutic development and continue to promote new ways to target AML cells. In addition, we describe clinical investigations that have led to FDA approval of new targeted AML therapies and ongoing clinical trials of novel therapies targeting AML survival pathways. We also describe the complexity of targeting leukemia stem cells (LSCs) as an approach to addressing relapse and remission in AML and targetable pathways that are unique to LSC survival. This comprehensive review details what we currently understand about the signaling pathways that support AML cell survival and the exceptional ways in which we disrupt them.

Targeting oxidative phosphorylation (OXPHOS) is a promising strategy to improve treatment outcomes of acute myeloid leukemia (AML) patients. IACS-010759 is a mitochondrial complex I inhibitor that has demonstrated preclinical antileukemic activity and is being tested in Phase I clinical trials. However, complex I deficiency has been reported to inhibit apoptotic cell death through prevention of cytochrome c release. Thus, combining IACS-010759 with a BH3 mimetic may overcome this mechanism of resistance leading to synergistic antileukemic activity against AML. In this study, we show that IACS-010759 and venetoclax synergistically induce apoptosis in OXPHOS-reliant AML cell lines and primary patient samples and cooperatively target leukemia progenitor cells. In a relatively OXPHOS-reliant AML cell line derived xenograft mouse model, IACS-010759 treatment significantly prolonged survival, which was further enhanced by treatment with IACS-010759 in combination with venetoclax. Consistent with our hypothesis, IACS-010759 treatment indeed retained cytochrome c in mitochondria, which was completely abolished by venetoclax, resulting in Bak/Bax- and caspase-dependent apoptosis. Our preclinical data provide a rationale for further development of the combination of IACS-010759 and venetoclax for the treatment of patients with AML.

No abstract

Recombination nodules (RNs) are closely correlated with crossing over, and, because they are observed by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs provide the highest-resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes. Using the maize inbred line KYS, we prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent. RN frequencies are closely correlated with both chiasma frequencies and SC length. The total length of the RN recombination map is about twofold shorter than that of most maize linkage maps, but there is good correspondence between the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe reduction of RNs at and near kinetochores. The frequency of RNs at knobs is either similar to or higher than the average frequency of RNs along the SCs. These RN maps represent an independent measure of crossing over along maize bivalents.

Summary: The purpose of this study is to demonstrate calcium alginate hydrogels as a system for in vitro radiobiological and metabolic studies of cancer cells. Previous studies have established calcium alginate as a versatile three-dimensional (3D) culturing system capable of generating areas of oxygen heterogeneity and modeling metabolic changes in vitro. Here, through dosimetry, clonogenic and viability assays, and pimonidazole staining, we demonstrate that alginate can model radiobiological responses that monolayer cultures do not simulate. Notably, alginate hydrogels with radii greater than 500 μm demonstrate hypoxic cores, while smaller hydrogels do not. The size of this hypoxic region correlates with hydrogel size and improved cell survival following radiation therapy. Hydrogels can also be utilized in hyperpolarized magnetic resonance spectroscopy and extracellular flux analysis. Alginate therefore offers a reproducible, consistent, and low-cost means for 3D culture of cancer cells for radiobiological studies that simulates important in vivo parameters such as regional hypoxia, as well as enables long-term culturing and in vitro metabolic studies.

Influenza is one of the most important infectious diseases in humans. The best way to prevent severe illness caused by influenza infection is vaccination. Cell culture-derived influenza vaccines are being considered in addition to the widely used egg-based system in order to support the increasing seasonal demand and to be prepared in case of a pandemic. Cell culture based systems offer increased safety, capacity, and flexibility with reduced downstream processing relative to embryonated eggs. We have previously reported a chick embryo cell line, termed PBS-12SF, that supports replication of human and avian influenza A viruses to high titers (>10 7 PFU/ml) without the need for exogenous proteases or serum proteins. Viral infections in cells are limited by the Interferon (IFN) response typified by production of type I IFNs that bind to the IFNa/b receptor and activate an antiviral state. In this study, we investigated how neutralizing the interferon (IFN) response in PBS-12SF cells, via shRNA-mediated knock-down of IFNAR1 mRNA expression, affects influenza virus production. We were successful in knocking down »90% of IFNAR1 protein expression by this method, resulting in a significant decrease in the response to recombinant chIFNa stimulation in PBS-12SF cells as shown by a reduction in expression of interferon-responsive genes when compared to control cells. Additionally; IFNAR1-knock-down cells displayed enhanced viral HA production and released more virus into cell culture supernatants than parental PBS-12SF cells.

No abstract

The 5-year survival rate for adult patients with acute myeloid leukemia (AML) treated with cytarabine-based chemotherapy remains less than 30%, due to drug resistance and disease relapse. Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was approved by the FDA in combination with low dose cytarabine or hypomethylating agents for treating newly diagnosed AML patients who are 75 years of age or older or for those who are unfit for standard chemotherapy, providing more treatment options for this group of patients. Although the response rate to these newly approved combination therapies is reported to be 70%, the median overall survival is only 10-18 months showing that the duration of response is limited. Therefore, novel therapeutic agents are in demand to enhance venetoclax activity against AML and to combat AML resistant to cytarabine-based chemotherapy. Cytarabine-resistant AML cells lead to relapse and rely on oxidative phosphorylation (OXPHOS) for survival. In addition, it has been reported that targeting OXPHOS can enhance venetoclax activity against preclinical models of AML. Thus, we hypothesize that OXPHOS suppressing agents would be good candidates to combine with and enhance venetoclax antileukemic activity against newly diagnosed AML and those with resistance to cytarabine. A novel isoflavone, ME-344, has been shown to suppress OXPHOS in cell lines derived from solid tumors by inhibiting Complex I of the electron transport chain. We hypothesized that combining ME-344 with venetoclax would result in synergistic antileukemic activity against AML. Consistent with our hypothesis, combining ME-344 with venetoclax resulted in synergistic induction of apoptosis in AML cell lines, including those with acquired cytarabine resistance. The combination of these two agents also resulted in synergistic antileukemic activity in one primary AML patient sample, as determined by MTT assay. The combination of ME-344 and venetoclax prolonged the median survival of MV4-11 leukemia- bearing NSGS mice by 37% (median survival of 48 days compared to 35 days for vehicle control treated mice, n=5 per arm, p<0.0001). This is in contrast to the venetoclax combination with cytarabine, which prolonged median survival of the same xenograft model by 7.5% (Luedtke et al., Signal Transduction and Targeted Therapy, 2020; 5:17). ME-344 alone (9-hour treatment) reduced basal mitochondrial respiration in AML cells by 10% prior to induction of apoptosis. When treated with ME-344 for 8-hours followed by combined ME-344 and venetoclax for an additional 1-hour, basal mitochondrial respiration was reduced by 18% (again prior to detection of apoptosis initiation). This sequential combination regimen also decreased the mitochondrial membrane potential (by JC-1 staining and flow cytometry analysis) when compared to untreated control and single treatment. Additionally, apoptosis induction by the combination of ME-344 and venetoclax or ME-344 alone was significantly enhanced when AML cells were forced to utilize OXPHOS by replacing glucose with galactose in the culture medium. Further investigation revealed that apoptosis induced by ME-344 was partially attenuated when Mcl-1 was overexpressed, Bak was knocked down, or caspase activation was inhibited. This suggests a mechanism that involves components of the intrinsic apoptosis pathway. Targeted metabolomics analyses of MV4-11 cells treated with ME-344 for 8 h revealed a significant reduction of essential metabolites involved in the de novo purine biosynthesis pathway, specifically AICAR (p=0.001) and IMP (p=0.004). Given the critical role of purine in cell proliferation and survival, suppression of purine biosynthesis by ME-344 may represent a novel mechanism underlying its enhancement on the antileukemic activity of venetoclax against AML. Interestingly, inhibition of this pathway by the purine biosynthesis inhibitor lometrexol, also synergistically enhanced apoptosis in AML cells induced by venetoclax. Taken together, these results suggest that ME-344 suppresses OXPHOS and the purine biosynthesis pathway to enhance the antileukemic activity of venetoclax against AML. Further in-depth mechanistic studies into the suppression of purine biosynthesis and OXPHOS, as well as studies of ME-344 and venetoclax against cytarabine-resistant AML in a mouse model are warranted. Disclosures Wiley: MEIPharma: Current Employment.