Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A.

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

Bioluminescence Assay of Lysine Deacylase Sirtuin Activity.

Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine towards maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple pathological conditions, including cancer. Measuring sirtuins' activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed 'SIRTify', a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc® luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine acylations and confirm the effects of sirtuin modulators. SIRTify effectively quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A.

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

SIRT5 IS A DRUGGABLE METABOLIC VULNERABILITY IN ACUTE MYELOID LEUKEMIA.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.

Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair.

Recent studies have implicated DNA polymerases θ (Pol θ) and ß (Pol ß) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

BCR-ABL1 tyrosine kinase inhibitor K0706 exhibits preclinical activity in Philadelphia chromosome-positive leukemia.

BCR-ABL1 tyrosine kinase inhibitors (TKIs) are the cornerstone of treatment in chronic myeloid leukemia. Although there are now four TKIs approved for use in the front-line setting, acquired TKI resistance via secondary kinase domain mutations remains a problem for patients. K0706 is a novel BCR-ABL1 TKI currently under clinical investigation with structural elements similar to those of ponatinib and dasatinib. In this article, we functionally characterize the anti-leukemic activity of K0706 using cell proliferation assays in conjunction with drug resistance screening. We provide details from molecular modeling to support our in vitro findings and additionally describe our limited clinical experience with this drug in two patients treated on trial. We demonstrate that although K0706 retains efficacy against a large spectrum of clinically relevant mutations, it does not appear to have activity against BCR-ABL1T315I. Early trial experience suggests excellent tolerability, which may positively affect the place of K0706 within the ever-expanding chronic myeloid leukemia treatment paradigm.

Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas.

Antibodies that bind CD47 on tumor cells and prevent interaction with SIRPα on phagocytes are active against multiple cancer types including T-cell lymphoma (TCL). Here we demonstrate that surface CD47 is heterogeneously expressed across primary TCLs, whereas major histocompatibility complex (MHC) class I, which can also suppress phagocytosis, is ubiquitous. Multiple monoclonal antibodies (mAbs) that block CD47-SIRPα interaction promoted phagocytosis of TCL cells, which was enhanced by cotreatment with antibodies targeting MHC class I. Expression levels of surface CD47 and genes that modulate CD47 pyroglutamation did not correlate with the extent of phagocytosis induced by CD47 blockade in TCL lines. In vivo treatment of multiple human TCL patient-derived xenografts or an immunocompetent murine TCL model with a short course of anti-CD47 mAb markedly reduced lymphoma burden and extended survival. Depletion of macrophages reduced efficacy in vivo, whereas depletion of neutrophils had no effect. F(ab')2-only fragments of anti-CD47 antibodies failed to induce phagocytosis by human macrophages, indicating a requirement for Fc-Fcγ receptor interactions. In contrast, F(ab')2-only fragments increased phagocytosis by murine macrophages independent of SLAMF7-Mac-1 interaction. Full-length anti-CD47 mAbs also induced phagocytosis by Fcγ receptor-deficient murine macrophages. An immunoglobulin G1 anti-CD47 mAb induced phagocytosis and natural killer cell-mediated cytotoxicity of TCL cells that was augmented by cotreatment with mogamulizumab, an anti-CCR4 mAb, or a mAb blocking MHC class I. These studies help explain the disparate activity of monotherapy with agents that block CD47 in murine models compared with patients. They also have direct translational implications for the deployment of anti-CD47 mAbs alone or in combination.

Mechanisms of Lymphoma Clearance Induced by High-Dose Alkylating Agents.

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a "super-phagocytic" subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. SIGNIFICANCE: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress-dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.See related commentary by Duval and De Palma, p. 834.This article is highlighted in the In This Issue feature, p. 813.

Biomarker-driven strategy for MCL1 inhibition in T-cell lymphomas.

There is a pressing need for more effective therapies to treat patients with T-cell lymphomas (TCLs), including first-line approaches that increase the response rate to cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) chemotherapy. We characterized the mitochondrial apoptosis pathway in cell lines and patient-derived xenograft (PDX) models of TCL and assessed the in vitro efficacy of BH3 mimetics, including the BCL2 inhibitor venetoclax, the BCL2/BCL-xL inhibitor navitoclax, and the novel MCL1 inhibitor AZD5991. The abundance of antiapoptotic BCL2 family members based on immunoblotting or RNA transcript levels correlated poorly with the activity of BH3 mimetics. In contrast, the functional approach BH3 profiling reliably predicted sensitivity to BH3 mimetics in vitro and in vivo. We used BH3 profiling to select TCL PDX that were dependent on MCL1. Mice xenografted with these PDX and treated with AZD5991 had markedly improved survival. The combination of AZD5991 and CHOP achieved synergy based on survival improvement beyond a mathematical "sum of benefits" model. Thus, MCL1 inhibition is a promising strategy as both a single agent and in combination with chemotherapy for patients with TCL and functional dependence on MCL1.

Parp3 promotes long-range end joining in murine cells.

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

Anti-CD37 chimeric antigen receptor T cells are active against B- and T-cell lymphomas.

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models.

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.