ABSTRACT:Perturbations in intermediary metabolism contribute to the pathogenesis of acute myeloid leukemia (AML) and can produce therapeutically actionable dependencies. Here, we probed whether α-ketoglutarate (αKG) metabolism represents a specific vulnerability in AML. Using functional genomics, metabolomics, and mouse models, we identified the αKG dehydrogenase complex, which catalyzes the conversion of αKG to succinyl coenzyme A, as a molecular dependency across multiple models of adverse-risk AML. Inhibition of 2-oxoglutarate dehydrogenase (OGDH), the E1 subunit of the αKG dehydrogenase complex, impaired AML progression and drove differentiation. Mechanistically, hindrance of αKG flux through the tricarboxylic acid (TCA) cycle resulted in rapid exhaustion of aspartate pools and blockade of de novo nucleotide biosynthesis, whereas cellular bioenergetics was largely preserved. Additionally, increased αKG levels after OGDH inhibition affected the biosynthesis of other critical amino acids. Thus, this work has identified a previously undescribed, functional link between certain TCA cycle components and nucleotide biosynthesis enzymes across AML. This metabolic node may serve as a cancer-specific vulnerability, amenable to therapeutic targeting in AML and perhaps in other cancers with similar metabolic wiring.

Although cell-fate specification is generally attributed to transcriptional regulation, emerging data also indicate a role for molecules linked with intermediary metabolism. For example, α-ketoglutarate (αKG), which fuels energy production and biosynthetic pathways in the tricarboxylic acid (TCA) cycle, is also a co-factor for chromatin-modifying enzymes 1–3 . Nevertheless, whether TCA-cycle metabolites regulate cell fate during tissue homeostasis and regeneration remains unclear. Here we show that TCA-cycle enzymes are expressed in the intestine in a heterogeneous manner, with components of the αKG dehydrogenase complex 4–6 upregulated in the absorptive lineage and downregulated in the secretory lineage. Using genetically modified mouse models and organoids, we reveal that 2-oxoglutarate dehydrogenase (OGDH), the enzymatic subunit of the αKG dehydrogenase complex, has a dual, lineage-specific role. In the absorptive lineage, OGDH is upregulated by HNF4 transcription factors to maintain the bioenergetic and biosynthetic needs of enterocytes. In the secretory lineage, OGDH is downregulated through a process that, when modelled, increases the levels of αKG and stimulates the differentiation of secretory cells. Consistent with this, in mouse models of colitis with impaired differentiation and maturation of secretory cells, inhibition of OGDH or supplementation with αKG reversed these impairments and promoted tissue healing. Hence, OGDH dependency is lineage-specific, and its regulation helps to direct cell fate, offering insights for targeted therapies in regenerative medicine.

Acute myeloid leukemia is the most common form of leukemia and can present with a wide variety of signs and symptoms. This article presents a case of a middle-aged male who presented with ongoing upper respiratory cold-like symptoms and was then found to be severely pancytopenic. A diagnosis of acute myeloid leukemia was made after a bone marrow biopsy, and the patient underwent induction chemotherapy. This article brings to light the uncommon diagnosis of acute myeloid leukemia, from a common presentation, a common cold. Additionally, it discusses the initial workup and diagnostic process of acute myeloid leukemia, risk stratification, and a basic treatment algorithm.

Metabolic rewiring and cellular reprogramming are trademarks of neoplastic initiation and progression in acute myeloid leukemia (AML). Metabolic alteration in leukemic cells is often genotype specific, with associated changes in epigenetic and functional factors resulting in the downstream upregulation or facilitation of oncogenic pathways. Targeting abnormal or disease-sustaining metabolic activities in AML provides a wide range of therapeutic opportunities, ideally with enhanced therapeutic windows and robust clinical efficacy. This review highlights the dysregulation of amino acid, nucleotide, lipid, and carbohydrate metabolism in AML; explores the role of key vitamins and enzymes that regulate these processes; and provides an overview of metabolism-directed therapies currently in use or development.

Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.

Background: Over 300 somatic molecular variants in hematologic diseases are either specified as diagnostic criteria in the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues, recognized as potentially actionable biomarkers in the National Comprehensive Cancer Network (NCCN) compendia, or supported by published well-powered clinical studies. Moreover, new molecular alterations with potential clinical implications in hematologic disease are continuously emerging in the scientific literature. These have critical use for a wide spectrum of clinicians, including hematopathologists who diagnose patient-specific hematologic malignancies, heme-oncologists who direct patient care, and clinical trial nurses who assist patients in finding appropriate clinical trials. Importantly, the utility of this information critically depends on the clinician's ability to interpret the significance of variants in a point-of-care setting. Therefore, there is an urgent and unmet need for a clinical decision support system that 1) distills the clinical implications associated with molecular alterations into a standardized and easily interpretable format and 2) democratizes access of this information to all members of the heme-oncology community. Methods: OncoKB is an established expert-guided precision oncology knowledge base that annotates the oncogenic effect and therapeutic implications of somatic molecular alterations (Chakravarty, D. et al., JCOPO, 2017). Previously, OncoKB was focused primarily on solid tumor mutation annotation. Recently, we expanded OncoKB to include alterations in hematologic malignancies. The heme-specific annotation efforts were guided by heme-oncology and hematopathology physician scientists at Memorial Sloan Kettering (MSK). Supplementing the previously published therapeutic levels of evidence (Fig. 1a), we further added level of evidence systems for diagnostic and prognostic implications (Fig. 1b, c). These three sets of evidence levels are consistent with the criteria set forth by the joint consensus of the ASCO/CAP/AMP guidelines (Li, MM. et al., J Mol Diagn, 2017). We assigned the newly curated heme-specific molecular alterations with diagnostic, prognostic or therapeutic levels of evidence, when applicable. Finally, we annotated and analyzed 1569 hematologic tumor samples from the AACR Project GENIE (release 6.1) with these levels of evidence. Results: In addition to alterations with both solid and heme clinical implications already curated in OncoKB, we annotated 288 unique heme-specific mutations, fusions, and copy number alterations in 156 newly curated cancer-associated genes. Based on MSK-expert consensus, the WHO and NCCN guidelines, and the scientific literature, we identified a total of 192 alterations with unique diagnostic levels of evidence, 65 alterations with unique prognostic levels of evidence and 55 alterations with unique therapeutic levels of evidence across 13 major hematologic tumor types (Fig. 2). To test the utility of OncoKB, we annotated all genomic events in 1569 heme cancer samples in 89 hematologic malignancies in the AACR GENIE cohort (V6.1) (Fig. 3a). Thirty-eight percent of samples harbored at least one potentially actionable alteration, and 8% were predictive of clinical benefit from an FDA-approved drug (Fig. 3b). Conclusions: OncoKB heme data is publicly available both through the web resource http://oncokb.org and through incorporation into the cBioPortal for Cancer Genomics. Heme-specific molecular alterations are used to make an accurate diagnosis, inform prognosis, optimize the use of stem cell transplant, and to link patients with the optimal mechanism-based therapies in the clinical trial setting and in routine clinical practice. This is the first study to annotate and analyze actionability of heme samples. In this proof-of-principle study, we demonstrate the ability to annotate clinical samples with their diagnostic, prognostic and therapeutic implications in a point-of-care setting. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Ho:Invivoscribe, Inc.: Honoraria. Knorr:Fate Therapeutics: Patents & Royalties. LaFave:Epizyme: Patents & Royalties. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Berger:Roche: Consultancy. Solit:Pfizer: Consultancy; Lilly Oncology: Honoraria; Vivideon Therapeutics: Consultancy; Loxo Oncology: Consultancy, Equity Ownership; Illumina: Consultancy. Dogan:Celgene: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy; Roche: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Gilead: Consultancy; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding.

Ubiquitin-mediated degradation of an inhibitor of noncanonical NF-κB signaling promotes cell survival.

Fbxw7α is a member of the F-box family of proteins, which function as the substrate-targeting subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Using differential purifications and mass spectrometry, we identified p100, an inhibitor of NF-κB signalling, as an interactor of Fbxw7α. p100 is constitutively targeted in the nucleus for proteasomal degradation by Fbxw7α, which recognizes a conserved motif phosphorylated by GSK3. Efficient activation of non-canonical NF-κB signalling is dependent on the elimination of nuclear p100 through either degradation by Fbxw7α or exclusion by a newly identified nuclear export signal in the carboxy terminus of p100. Expression of a stable p100 mutant, expression of a constitutively nuclear p100 mutant, Fbxw7α silencing or inhibition of GSK3 in multiple myeloma cells with constitutive non-canonical NF-κB activity results in apoptosis both in cell systems and xenotransplant models. Thus, in multiple myeloma, Fbxw7α and GSK3 function as pro-survival factors through the control of p100 degradation.

RANTES (CCL5) is a chemokine implicated in many human diseases. We previously showed that the transcription factor Kruppel-like factor 13 (KLF13) controls the late (3-5 days after activation) expression of RANTES in T lymphocytes and that KLF13 itself is translationally regulated through the 5'-untranslated region of its mRNA. Here, we show that KLF13 levels are further regulated by ubiquitination and degradation. KLF13 protein is undetectable in resting human T lymphocytes, but treatment with either proteosomal or lysosomal inhibitors increases KLF13 protein levels. Glycogen synthase kinase 3β (GSK3β)-mediated phosphorylation of KLF13 triggers the ubiquitination of KLF13 by the E3 ligase Fbw7γ, resulting in KLF13 protein degradation. Knockdown of either Fbw7γ or GSK3β by small interfering RNA increases KLF13 expression in resting human T lymphocytes. In contrast, in murine T lymphocytes, KLF13 protein is abundant because of the absence of Fbw7γ. Treatment of unactivated human lymphocytes with lysosomal inhibitors stabilizes KLF13 protein, resulting in an increase of RANTES mRNA and protein. Taken together, these studies found that tightly regulated control of both synthesis and degradation allows rapid changes in the level of KLF13 in human T lymphocytes.

On the basis of differential analysis of affinity purifications by mass spectrometry, we identified the nuclear factor κB (NF-κB) protein p100 (NF-κB2) as an interactor of the F-box protein FBXW7α. The NF-κB pathway is important for cell growth, differentiation, and survival. p100, which shuttles between the cytoplasm and nucleus, functions as the primary inhibitor of the noncanonical NF-κB pathway by sequestering NF-κB heterodimers in the cytoplasm. In the absence of NF-κB stimulation, the nuclear pool of p100 is constitutively targeted for degradation by FBXW7α, which recognizes a conserved motif that is phosphorylated by glycogen synthase kinase 3 (GSK3). Efficient activation of noncanonical NF-κB signaling depends on the clearance of nuclear p100, either through FBXW7α-mediated degradation or nuclear export mediated by a signal in the C terminus of p100. Upon prolonged stimulation of the NF-κB pathway, p100 is stabilized and retained in the nucleus, contributing to the cessation of noncanonical NF-κB signaling. The molecular mechanism of p100 degradation has implications in multiple myeloma, a disease with constitutive activation of the noncanonical NF-κB pathway. Accordingly, expression of a stable p100 mutant, FBXW7α depletion, or chemical inhibition of GSK3 in multiple myeloma cells results in cell death in vitro and in a xenotransplant model. Thus, the FBXW7α-dependent degradation of p100 functions as a prosurvival mechanism through control of NF-κB activity.

During mitosis, eukaryotic cells have to properly align their chromosomes. Only after the kinetochore of each chromosome is attached to a polar microtubule can a cell satisfy the ‘spindle assembly checkpoint’, which prevents the mis-segregation of chromosomes. Failure to correctly segregate chromosomes before cell division might contribute to chromosome instability and tumorigenesis. to counteract chromosome aberrations, the cell initiates the apoptotic programme. it has become clear through the use of microtubule-poisoning, chemotherapeutic agents—such as paclitaxel and vincristine—that prolonged activation of the spindle checkpoint can induce mitotic arrest and, subsequently, programmed cell death. the molecular mechanisms responsible for initiating apoptosis during mitotic arrest have remained poorly defined. two recent papers in Nature (inuzuka et al, 2011; Wertz et al, 2011) and a report published by the clarke group last year in The EMBO Journal (Harley et al, 2010) highlight the destruction of McL1 during prolonged mitotic arrest and shed light on the mechanisms of apoptosis induction. Myeloid cell leukaemia 1 (McL1) is an anti-apoptotic member of the B-cell lymphoma 2 (BcL2) family of proteins. McL1, like BcL2 and BcLxL, prevents the downstream activation of BaX and BaK, which are responsible for mitochondrial outermembrane perme abilization, initiation of the caspase cascade and induction of apoptosis (youle & Strasser, 2008). ubiquitination and proteolysis of McL1 have been reported, but a mechanism for McL1 degradation following spindle checkpoint activation remains unknown. Now, the studies referenced above suggest that degradation of McL1 during prolonged mitotic arrest is essential for the induction of apoptosis. given its prominent role in driving the cell cycle, as well as in safeguarding the fidelity of this process, it is not surprising that the ubiquitin-proteasome system (upS) has a key role in dictating the activation of the intrinsic apoptotic pathway in cells arrested in mitosis. However, it is surprising that two E3 ubiquitin ligase complexes simultaneously facilitate this degradation event. Harley and colleagues describe the regulation of McL1 by apc/ccdc20 (anaphasepromoting complex/cyclosome and its activator cdc20). this multi-subunit riNg E3 ubiquitin ligase is active in mitosis, and ubiquitinates substrates such as securin and cyclin B, thereby allowing progression into anaphase. in their report, Harley and coworkers (2010) demonstrate a cdk1/cyclin-Bmediated, site-specific phosphorylation (thr 92 in humans) of McL1 upon mitotic arrest, followed by its proteolytic de struction by apc/ccdc20. thus, like the sand of an hourglass flipped at each entry into mitosis, the level of McL1 steadily decreases. if time ‘runs out’ due to a prolonged mitotic arrest (that is, if McL1 is completely destroyed), then apoptosis is initiated (Fig 1a). Both phosphorylation at thr 92 and the presence of a conserved destruction or ‘D’-box motif (a characteristic of apc/c substrates) are required for McL1 proteolysis, although the precise role of phosphorylation in promoting degradation remains unclear. interestingly, the stability of McL1 in asynchronous cells seems to be unaffected when the ability of apc/ccdc20 to target McL1 is compromised by knockdown of cdc20, or when phosphorylation at thr 92 is ablated. although the spindle assembly checkpoint is believed to inhibit apc/ccdc20 activity, the degradation of some targets, such as the cDK-inhibitor p21 and cyclin a, is not affected. consequently, it is possible that McL1 can be destroyed through cdc20 during mitotic arrest. More recently, in two reports in Nature (inuzuka et al, 2011; Wertz et al, 2011), it is shown that McL1 interacts with another E3 ubiquitin ligase, ScFFbxw7. Similarly to the apc/c, the ScF (Skp1/cul1/F-box protein) is a multi-subunit, riNg E3 ubiquitin ligase. the F-box protein provides the specificity for target recognition, often by using specific interaction domains to bind to substrates. in the case of Fbxw7 (also known as Fbw7 and hcdc4), a series of WD40 domains

Multiple myeloma (MM) is the second most common hematologic malignancy and is considered incurable. The development of effective therapeutics, including alkylating agents, proteasome inhibitors (PI), and other targeted therapies used alone or in combination has improved patient outcomes. Recent work on signaling pathways in MM, including the NF-κB, Wnt/β-Catenin and Ras/MAPK pathways, suggests a role for GSK-3 inhibition in MM. A determination of the optimal ordering and timing of delivery of ionizing radiation (IR), PI, and other therapies in MM cell lines may be used to guide clinical trial design. We analyzed three MM cells lines: ARP-1, KMS-11 (p53 mut) and MM.1r (p53 wt). Cells were grown under standard tissue culture conditions in suspension culture. Cell viability assays were performed in 96-well plates using MTT reagents. Viability assays took place 72 hrs after treatment with drug or IR for 1 - 4 h and read with a SoftMax fluorescent reader (Molecular Devices). Dose-response curves were generated using appropriate serial dilution techniques. Assays were performed in triplicate to generate mean and standard deviations. The method of Chou and Talalay was used to assess interactions using a combination index (CI) to measure the degree of synergism and/or antagonism in dose-effect relationships. Cell lines were analyzed to determine the IC50 concentrations of the following treatments: bortezomib (proteasome inhibitor), bendamustine and cyclophosphamide (alkylators), dexamethasone, and GSK-3B inhibitor IX. The cell lines were sensitive to bortezomib treatment with an IC50 between 1 - 10 nM. ARP-1 was moderately sensitive to bendamustine, but resistant to doses of cyclophosphamide and dexamethasone up to 100 μM, whereas KMS-11 and MM.1r were resistant to bendamustine, cyclophosphamide and dexamethasone. The GSK3 inhibitor resulted in decreased viability of all three cell lines with an IC50 of between 1 - 5 μM. The ARP-1 line was sensitive to IR, whereas KMS-11 and MM-1r only had a 15 - 20 percent decline in MTT activity following IR up to 10 Gy. Delivery of bortezomib in ARP-1 cells either 2 h prior to or 2 h after IR demonstrated additive cell killing, but without significant synergy/antagonism (CI ≈ 1). The response of several MM cell lines to IR, PI and other therapeutics was characterized. Preliminary analysis suggests that GSK3 inhibition may be a novel, effective therapeutic strategy in MM. Although ARP-1 cells are sensitive to both bortezomib and IR, the combination results in additive killing rather than a true synergistic effect at the drug levels tested. This may reflect the limits of the MTT assay system used in these experiments. This approach may be hypothesis-generating for clinical trial design in MM.

Evidence for the destruction of the anti‐apoptotic protein MCL1 during prolonged mitotic arrest comes from three papers, one in The EMBO Journal and two in Nature , thus shedding light on the mechanism of apoptosis induction under these conditions.