Superoxide dismutases (SOD) are essential enzymes that eliminate superoxide radical (O2-) and thus protect cells from damage induced by free radicals. The active O2- production and low SOD activity in cancer cells may render the malignant cells highly dependent on SOD for survival and sensitive to inhibition of SOD. Here we report that certain oestrogen derivatives selectively kill human leukaemia cells but not normal lymphocytes. Using complementary DNA microarray and biochemical approaches, we identify SOD as a target of this drug action and show that chemical modifications at the 2-carbon (2-OH, 2-OCH3) of the derivatives are essential for SOD inhibition and for apoptosis induction. Inhibition of SOD causes accumulation of cellular O2- and leads to free-radical-mediated damage to mitochondrial membranes, the release of cytochrome c from mitochondria and apoptosis of the cancer cells. Our results indicate that targeting SOD may be a promising approach to the selective killing of cancer cells, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.

FCR produced a high CR rate in previously untreated CLL. Most patients had no detectable disease on flow cytometry at the end of therapy. Time to treatment failure analysis showed that 69% of patients were projected to be failure free at 4 years (95% CI, 57% to 81%).

• FCR-treated chronic lymphocytic leukemia patients with mutated IGHV gene achieve long-term PFS, with a plateau on the PFS curve.• MRD-negativity posttreatment is highly predictive of longterm PFS, particularly in patients with mutated IGHV gene.Accurate identification of patients likely to achieve long-progression-free survival (PFS) after chemoimmunotherapy is essential given the availability of less toxic alternatives, such as ibrutinib. Fludarabine, cyclophosphamide, and rituximab (FCR) achieved a high response rate, but continued relapses were seen in initial reports. We reviewed the original 300 patient phase 2 FCR study to identify long-term disease-free survivors. Minimal residual disease (MRD) was assessed posttreatment by a polymerase chain reaction-based ligase chain reaction assay (sensitivity 0.01%). At the median follow-up of 12.8 years, PFS was 30.9% (median PFS, 6.4 years). The 12.8-year PFS was 53.9% for patients with mutated immunoglobulin heavy chain variable (IGHV) gene (IGHV-M) and 8.7% for patients with unmutated IGHV (IGHV-UM). 50.7% of patients with IGHV-M achieved MRD-negativity posttreatment; of these, PFS was 79.8% at 12.8 years. A plateau was seen on the PFS curve in patients with IGHV-M, with no relapses beyond 10.4 years in 42 patients (total follow-up 105.4 patient-years). On multivariable analysis, IGHV-UM (hazard ratio, 3.37 [2.18-5.21]; P < .001) and del(17p) by conventional karyotyping (hazard ratio, 7.96 [1.02-61.92]; P 5 .048) were significantly associated with inferior PFS. Fifteen patients with IGHV-M had 4-color MRD flow cytometry (sensitivity 0.01%) performed in peripheral blood, at a median of 12.8 years posttreatment (range, 9.5-14.7). All were MRD-negative. The high rate of very long-term PFS in patients with IGHV-M after FCR argues for the continued use of chemoimmunotherapy in this patient subgroup outside clinical trials; alternative strategies may be preferred in patients with IGHV-UM, to limit long-term toxicity. (Blood. 2016;127(3):303-309)

Tissue stromal cells interact with leukemia cells and profoundly affect their viability and drug sensitivity. Here we show a biochemical mechanism by which bone marrow stromal cells modulate the redox status of chronic lymphocytic leukemia (CLL) cells and promote cellular survival and drug resistance. Primary CLL cells from patients exhibit limited ability to transport cystine for glutathione (GSH) synthesis due to a low expression of Xc- transporter, while bone marrow stromal cells effectively import cystine and convert it to cysteine, which is then released into the microenvironment for uptake by CLL cells to promote GSH synthesis. The elevated GSH enhances leukemia cell survival and protects them from drug-induced cytotoxicity. Furthermore, disabling this protective mechanism significantly sensitizes CLL cells to drug treatment in stromal environment. This stromal-leukemia interaction is critical for CLL cell survival and represents a key biochemical pathway for effectively targeting leukemia cells to overcome drug resistance in vivo.

Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (ρ-) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.

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IntroductionWith the establishment of more effective treatments for patients with chronic lymphocytic leukemia (CLL) over the past decade, complete remissions are no longer the exception. 1 Despite these major improvements in CLL treatment, we still consider CLL an incurable disease, because patients generally relapse from minimal residual disease (MRD). 2 There is growing evidence suggesting that CLL cells are protected from conventional drugs in tissue microenvironments, such as the bone marrow and secondary lymphoid organs, with facilitation of residual disease that is drug resistant and ultimately paving the way to clonal evolution and relapses. The complex cellular and molecular contexts in the tissues, collectively referred to as the CLL microenvironment, provide signals for the expansion of the CLL clone and for primary drug resistance. This is largely dependent on direct contact between the malignant B cells and stromal cells, 3 and therefore has been designated as cell adhesion-mediated drug resistance. 4 Disrupting cross talk between leukemia cells and their milieu is an attractive novel but yet incompletely tested strategy for treating CLL. Appropriately, there is growing interest in understanding the biology of CLL-stroma cross talk to find ways to eliminate residual CLL cells that are "hiding" in stromal niches within the marrow and the lymphatic tissues.Importantly, once CLL cells are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma, they undergo spontaneous apoptosis, highlighting the importance of external signals from accessory cells. 5 Previous studies have shown that CLL cell cocultures with different adherent cell types, collectively referred to as stromal cells, induce leukemia cell survival, migration, and drug resistance. These stromal cells include mesenchymal marrow stromal cells (MSCs), 3,6,7 CD68 ϩ nurselike cells derived from monocytes, 7-10 and follicular dendritic cells. 11 Immunohistochemistry showed that in situ, ␣SMA ϩ mesenchymal stromal cells, 12 the in vivo counterpart of MSCs, are a dominant stromal cell population in the CLL microenvironment, which is in contrast to other B-cell lymphomas, particularly high-grade lymphomas, which harbor larger numbers of CD68 ϩ hemangiogenic cells. 12 MSCs regulate normal hematopoiesis by providing attachment sites and secreted or surface-bound growth factors that constitute the marrow microenvironment. 13 During B-cell development in the marrow, programmed cell death regulates B-cell homeostasis by diverting a large fraction of immature B cells into an apoptotic death pathway to eliminate functionless or potentially harmful cells. 14,15 Critical factors for the survival of selected B cells are interactions with MSCs in the marrow microenvironment, [16][17][18] expression of surface immunoglobulin molecules, and expression of apoptosis-regulatory proteins, such as Bcl-2. 19 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the For personal use only. on May 7,...

IntroductionChronic lymphocytic leukemia (CLL) is characterized by the gradual accumulation of small mature B cells, most of which are nonproliferating cells that display the T-cell marker CD5 in addition to the typical B-cell surface marker CD19. 1,2 High levels of the antiapoptotic B-cell lymphoma (Bcl-2) family proteins are expressed in most cases of CLL. This was correlated with resistance to therapy and may account for the prolonged survival of CLL cells. 3,4 Reduction in their levels in model systems and in CLL cells by antisense oligodeoxynucleotides or small interfering RNA (siRNA) causes cell death. [5][6][7] The purine nucleoside analog, fludarabine, is the most active single agent in the treatment of CLL. It induces higher response rates and longer progression-free survival than DNA alkylating agents such as chlorambucil. 8 Nevertheless, CLL almost uniformly progresses to refractory disease given sufficient time. 9 The underlying defects in apoptosis are likely to be the major contributors to therapeutic resistance. In addition, 40% to 50% of clinical resistance to fludarabine alone or in combination with alkylating agents is associated with mutation or deletion in the P53 gene. 10,11 Therefore, novel agents or strategies are needed to abrogate blocks to apoptosis in CLL, particularly in the treatment of patients who become refractory to primary therapy.Flavopiridol (NSC649890) is a semisynthetic flavonoid derived from an indigenous plant from India. 12 It has shown potent cytotoxicity on CLL cells in vitro [13][14][15] and promising activity in clinical trials. 16 Importantly, this proapoptotic activity of flavopiridol is independent of p53 function, 12,13 which makes it a potential drug to overcome resistance associated with p53 abnormalities. Flavopiridol inhibits cyclin-dependent kinases (CDKs) by competing with adenosine triphosphate (ATP) for the active site of each kinase. The activities of CDK1, CDK2, CDK4, CDK6, and CDK7 are inhibited with inhibitory concentration at 50% (IC 50 ) values in the range of 20 to 300 nM. 12 Consistent with this action, in growing cell populations flavopiridol blocks cell-cycle progression at the G 1 /S and G 2 /M boundaries. However, since most CLL cells are not actively cycling, the inhibition of cell-cycle-related CDKs is unlikely to be the major mechanism for its cytotoxicity. In seeking the mechanism of flavopiridol-induced apoptosis, Kitada et al observed that flavopiridol toxicity to CLL cells in vitro was associated with a decline of antiapoptotic proteins such as Bcl-2, myeloid-cell leukemia 1 (Mcl-1), X-linked inhibitor of apoptosis (XIAP), and Bcl-2-associated athanogene 1 (BAG-1). 14 However, the molecular basis for the observed down-regulation by flavopiridol remains to be determined.It is now known that flavopiridol decreases transcription by inhibiting CDK9 [17][18][19]20 which are responsible for the phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II, an activity essential for both transcriptional initiation ...