Obesity is associated with poorer outcome for many cancers. Previously, we observed that adipocytes protect acute lymphoblastic leukemia (ALL) cells from the anthracycline, daunorubicin (DNR). In the present study, it is determined whether adipocytes clear DNR from the tumor microenvironment (TME). Intracellular DNR concentrations were evaluated using fluorescence. DNR and its largely inactive metabolite, daunorubicinol, were analytically measured in media, cells, and tissues using liquid chromatography/mass spectrometry (LC/MS). Expression of DNR-metabolizing enzymes: aldo-keto reductases (AKR1A1, AKR1B1, AKR1C1, AKR1C2, AKR1C3, and AKR7A2) and carbonyl reductases (CBR1, CBR3) in human adipose tissue were queried using public databases, and directly measured by quantitative PCR (qPCR) and immunoblot. Adipose tissue AKR activity was measured by colorimetric assay. Adipocytes absorbed and efficiently metabolized DNR to daunorubicinol reducing its anti-leukemia effect in the local microenvironment. Murine studies confirmed adipose tissue conversion of DNR to daunorubicinol in vivo. Adipocytes expressed high levels of AKR and CBR isoenzymes that deactivate anthracyclines. Indeed, adipocyte protein levels of AKR1C1, AKR1C2, and AKR1C3 are higher than all other human non-cancerous cell types. To our knowledge, this is the first demonstration that adipocytes metabolize and inactivate a therapeutic drug. Adipocyte-mediated DNR metabolism reduces active drug concentration in the TME. These results could be clinically important for adipocyte-rich cancer microenvironments such as omentum, breast, and marrow. Since AKR and CBR enzymes metabolize several drugs, and can be expressed at higher levels in obese individuals, this proof-of-principle finding has important implications across many diseases.

fWe previously showed that the orphan nuclear receptor Nur77 (Nr4a1) plays an important role in the regulation of glucose homeostasis and oxidative metabolism in skeletal muscle. Here, we show using both gain-and loss-of-function models that Nur77 is also a regulator of muscle growth in mice. Transgenic expression of Nur77 in skeletal muscle in mice led to increases in myofiber size. Conversely, mice with global or muscle-specific deficiency in Nur77 exhibited reduced muscle mass and myofiber size. In contrast to Nur77 deficiency, deletion of the highly related nuclear receptor NOR1 (Nr4a3) had minimal effect on muscle mass and myofiber size. We further show that Nur77 mediates its effects on muscle size by orchestrating transcriptional programs that favor muscle growth, including the induction of insulin-like growth factor 1 (IGF1), as well as concomitant downregulation of growth-inhibitory genes, including myostatin, Fbxo32 (MAFbx), and Trim63 (MuRF1). Nur77-mediated increase in IGF1 led to activation of the Akt-mTOR-S6K cascade and the inhibition of FoxO3a activity. The dependence of Nur77 on IGF1 was recapitulated in primary myoblasts, establishing this as a cell-autonomous effect. Collectively, our findings identify Nur77 as a novel regulator of myofiber size and a potential transcriptional link between cellular metabolism and muscle growth. Skeletal muscle serves indelible roles in mediating locomotion and postural tone, as well as in the maintenance of energy homeostasis. Muscle wasting is commonly observed in patients with primary neuromuscular pathologies as well as in those with cancer cachexia. Much underappreciated, however, is the vast number of people who develop muscle atrophy as a comorbidity of aging, disuse, diabetes, heart failure, and chronic inflammatory illnesses. Muscle loss not only impairs the activities of daily living but also increases the risk of developing diabetes and of mortality (1-4). Current approaches of mitigating muscle loss-nutritional support and exercise-may be insufficient or infeasible in certain patient populations. Understanding the fundamental signaling pathways that control muscle mass is thus paramount to the development of novel therapies.Maintenance of muscle mass in the adult animal depends largely on the balance of signals that favor growth or atrophy. Environmental cues, including protein excess, growth factors, physical exercise, and ␤-adrenergic stimulation, activate a complex array of overlapping signaling pathways affecting muscle homeostasis (5, 6). The most well known pathway, the insulin-like growth factor 1 (IGF1)-Akt-mTOR cascade, promotes protein synthesis through concurrent regulation of multiple components of the translational machinery. Muscle differentiation and growth are also modulated by mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38, which can be activated by calcium as well as calciumindependent pathways (7-11). PGC1␣4 has also been shown to be a mediator of exercise-induced...

Muscle atrophy is a prevalent condition in illness and aging. Identifying novel pathways that control muscle mass may lead to therapeutic advancement. We previously identified Nur77 as a transcriptional regulator of glycolysis in skeletal muscle. More recently, we showed that Nur77 expression also controls myofiber size in mice. It was unknown, however, whether Nur77’s regulation of muscle size begins during developmental myogenesis or only in adulthood. To determine the importance of Nur77 throughout muscle growth, we examined myofiber size at E18.5, 3 weeks postnatal age, and in young adult mice. Using the global Nur77-/- mice, we showed that Nur77 deficiency reduced myofiber size as early as E18.5. The reduction in myofiber size became more pronounced by 3 weeks of age. We observed comparable reduction in myofiber size in young myofiber-specific Nur77-knockout mice. These findings suggest that Nur77’s effect on muscle growth is intrinsic to its expression in differentiating myofibers, and not dependent on its expression in myogenic stem cells. To determine the importance of Nur77 expression in muscle accretion in mature mice, we generated an inducible-, muscle-specific, Nur77-deficient mouse model. We demonstrated that tamoxifen-induced deletion of Nur77 in 3-month-old mice reduced myofiber size. This change was accompanied by increased activity of Smad2 and FoxO3, two negative regulators of muscle mass. The role of Nur77 in muscle growth was further elaborated in the cardiotoxin-induced muscle regeneration model. Compared to wildtype mice, regenerated myofibers were smaller in Nur77-/- mice. However, when normalized to saline-injected muscle, the recovery of sarcoplasmic area was comparable between Nur77-/- and wildtype mice. These findings suggest that Nur77 deficiency compromises myofiber growth, but not the regenerative capacity of myogenic progenitor cells. Collectively, the findings presented here demonstrate Nur77 as an important regulator of muscle growth both during prenatal and postnatal myogenesis.

Obesity is associated with poorer outcome from many cancers, including childhood acute lymphoblastic leukemia (ALL). We have previously shown that adipocytes protect ALL cells from the anthracycline, daunorubicin (DNR). We therefore investigated whether adipocytes sequester and/or metabolize DNR in the ALL microenvironment. Using fluorescence and LC/MS measures, we demonstrated that adipocytes absorb DNR, reducing the intracellular DNR concentration in co-cultured BV173 ALL cells (after 48 hours, median fluorescent intensity of ALL cultured with adipocytes was 1.7±1.0 vs. 5.0±1.7 of those cultured alone, p<0.01). Mouse adipocytes convert DNR to the less active metabolite, daunorubicinol (DNR-ol); over 48 hours, media DNR decreased to 0.1±0.2 (vs. 24.8±8.76 ng/mL in control wells). At the same time, DNR-ol increased to 15.0±1.9 (vs. 0.7±0.6 in control wells; both p<0.05). Similar conversion of DNR to DNR-ol was observed in both mouse adipose explants and human adipose tissue biopsy samples ex vivo. qPCR confirmed human subcutaneous adipose tissue expresses several enzymes capable of metabolizing DNR, including AKR1A1, 1B1, 1C1, 1C2, 1C3, 7A2, and CBR1 and 3 (expression ranged between 20 and 195% β-actin). Using immunohistochemistry, we confirmed expression of AKR1C1, 1C2, and 1C3 in bone marrow adipocytes of children during the first month of treatment for ALL. Finally, two hours after an intravenous dose of DNR in mice, we found that the DNR-ol to DNR ratio was higher in subcutaneous (0.60±0.26) and omental (0.55±0.21) adipose than in white blood cells (0.16±0.11), bone marrow (undetectable DNR-ol), and spleen (undetectable DNR-ol). Together, these data demonstrate that adipocytes sequester and inactivate DNR, likely due to their expression of AKR and CBR enzymes. These findings uncover a novel and important mechanism which could promote local anthracycline resistance by cancer cells in microenvironment rich in adipocytes, such as bone marrow, omentum, and breast. Citation Format: Xia Sheng, Jean-Hugues Parmentier, Jonathan Tucci, Hua Pei, Omar Cortez-Toledo, Christina Dieli-Conwright, Matthew Oberley, Michael Neely, Etan Orgel, Stan Louie, Steven D. Mittelman. Adipocytes sequester and metabolize daunorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2017-2960

<p>Primer sequences used for rtPCR.</p>