ALBERT

Description: We have designed in vitro assays to investigate the possible association between apoptosis and chemotherapeutic sensitivity in acute myeloid leukemias (AMLs). Consistent low levels of spontaneous apoptosis were observed in myeloid cells from normal bone marrow samples, while untreated cells collected from 56 de novo AML patients showed variable apoptosis. Control myeloid cells showed increased apoptosis after in vitro treatments with daunomycin (DNR), cytosine arabinoside (ARA-C), or gamma irradiation (RAD). Most AML samples showed less treatment-associated apoptosis, suggesting that apoptosis responses to therapeutic agents may be frequently attenuated in AML. Certain cytogenetic abnormalities common in AML may affect apoptosis, as acute promyelocytic leukemia (APL) samples with t(15; 17) karyotypes showed consistently low levels of spontaneous and treatment-associated apoptosis. Apoptosis assays may provide unique functional subtyping of AMLs, as other common cytogenetic subsets showed variable apoptosis. Altered function of two well-characterized regulators of apoptosis, BCL-2 and p53, was not entirely responsible for this variability. A genomic p53 mutation was found in only one AML sample. All samples that demonstrated the highest BCL-2–positive cell fractions showed low apoptosis, but reduced apoptosis was seen in both the presence and absence of BCL-2 overexpression. Finally, data from matched diagnosis and relapse sample pairs suggest that neither further reduced apoptosis nor additional BCL-2 overexpression is necessarily associated with disease progression.

Description: We have designed in vitro assays to investigate the possible association between apoptosis and chemotherapeutic sensitivity in acute myeloid leukemias (AMLs). Consistent low levels of spontaneous apoptosis were observed in myeloid cells from normal bone marrow samples, while untreated cells collected from 56 de novo AML patients showed variable apoptosis. Control myeloid cells showed increased apoptosis after in vitro treatments with daunomycin (DNR), cytosine arabinoside (ARA-C), or gamma irradiation (RAD). Most AML samples showed less treatment-associated apoptosis, suggesting that apoptosis responses to therapeutic agents may be frequently attenuated in AML. Certain cytogenetic abnormalities common in AML may affect apoptosis, as acute promyelocytic leukemia (APL) samples with t(15; 17) karyotypes showed consistently low levels of spontaneous and treatment-associated apoptosis. Apoptosis assays may provide unique functional subtyping of AMLs, as other common cytogenetic subsets showed variable apoptosis. Altered function of two well-characterized regulators of apoptosis, BCL-2 and p53, was not entirely responsible for this variability. A genomic p53 mutation was found in only one AML sample. All samples that demonstrated the highest BCL-2–positive cell fractions showed low apoptosis, but reduced apoptosis was seen in both the presence and absence of BCL-2 overexpression. Finally, data from matched diagnosis and relapse sample pairs suggest that neither further reduced apoptosis nor additional BCL-2 overexpression is necessarily associated with disease progression.

Description: The antibody-targeted therapeutic, gemtuzumab ozogamicin (GO, Mylotarg), is approved for treatment of relapsed acute myeloid leukemia (AML). We previously showed that AML blasts from GO refractory patients frequently express the drug transporters P-glycoprotein (Pgp) and/or multidrug resistance protein (MRP). We also previously reported that inhibition of drug transport by the Pgp modulator, cyclosporine A (CSA), can increase GO sensitivity in Pgp+ AML cells and that the peripheral benzodiazepine receptor ligand, PK11195, sensitizes AML cells to standard chemotherapeutics both by inhibiting Pgp-mediated efflux and by promoting mitochondrial apoptosis. We now show that PK11195 also can overcome multiple resistance mechanisms to increase GO sensitivity in AML cells, including resistance associated with expression of drug transporters and/or antiapoptotic proteins. PK11195 substantially increases GO cytotoxicity in AML cells from many different cell lines and primary patient samples, often more effectively than CSA. We also show that PK11195 is nontoxic in NOD/SCID mice and can sensitize xenografted human AML cells to GO. Since PK11195 is well tolerated in humans as a single agent, its further study as a multifunctional chemosensitizer for anti-AML therapies, including GO-based therapies, is warranted. (Blood. 2004;103:4276-4284)

Description: The peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux to chemosensitize cancer cells at least as well or better than the Pgp modulator, cyclosporine A (CSA). We now show that PK11195 broadly inhibits adenosine triphosphate (ATP)-binding cassette (ABC) transporters in hematologic cancer cell lines and primary leukemia-cell samples, including multidrug resistance protein (MRP), breast cancer resistance protein (BCRP), and/or Pgp. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but showed that pBR expression is unnecessary to PK11195-mediated efflux inhibition. PK11195 binds plasma-membrane sites in Pgp-expressing cells, stimulates Pgp-associated adenosine triphosphatase (ATPase) activity, and causes conformational changes in Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bind noncompetitively in Pgp-expressing cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites. Importantly, PK11195 concentrations that were effective in these in vitro assays can be safely achieved in patients. Because PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism and broadly blocks drug efflux by an apparently pBR-independent, ABC transporter-dependent mechanism, PK11195 may be a useful clinical chemosensitizer in cancer patients.

Description: Following exposure to cytotoxic agents, acute myeloid leukemia (AML) blasts elevate cellular cholesterol in a defensive adaptation that increases chemoresistance, but blockade of HMG-CoA reductase with statins restores chemosensitivity in vitro. This phase 1 study evaluated adding pravastatin (PV) (40-1680 mg/day, days 1-8) to idarubicin (Ida) ([12 mg/(M2 · day), days 4-6]) + high-dose cytarabine (Ara-C; HDAC) [1.5 g/(M2 · day) by CI, days 4-7] in 15 newly diagnosed and 22 salvage patients with unfavorable (n = 26) or intermediate (n = 10) prognosis cytogenetics. Compared with historical experience with Ida-HDAC, the duration of neutropenia and throbmbocytopenia and the toxicity profile were unaffected by the addition of PV. During PV loading (day 0-4) serum triglyceride and total and LDL cholesterol levels decreased in nearly all patients. Pharmacokinetic studies demonstrated higher and more sustained serum PV levels with PV doses above 1280 mg/day. CR/CRp was obtained in 11 of 15 new patients, including 8 of 10 with unfavorable cytogenetics, and 9 of 22 salvage patients. An MTD for PV + Ida-HDAC was not reached. Addition of PV to Ida-HDAC was safe, and the encouraging response rates support conducting further trials evaluating the effect of cholesterol modulation on response in AML.

Description: Cholesterol levels are abnormally increased in many acute myeloid leukemia (AML) samples exposed in vitro to chemotherapy. Blocking these acute cholesterol responses selectively sensitizes AML cells to therapeutics. Thus, defining the molecular mechanisms by which AML cells accomplish these protective cholesterol increments might elucidate novel therapeutic targets. We now report that the levels of mRNAs encoding the cholesterol synthesis-regulating enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the cholesterol-importing low-density lipoprotein (LDL) receptor were both increased by daunorubicin (DNR) or cytarabine (ARA-C) treatments in almost three fourths of cultured AML samples. However, less than one third of AML samples significantly increased LDL accumulation during drug treatments, suggesting that de novo synthesis is the primary mechanism by which most AML cells increase cholesterol levels during drug exposures. LDL increments were not correlated with cholesterol increments in ARA-C–treated AML samples. However, LDL and cholesterol increments did correlate in DNR-treated AML samples where they were measured, suggesting that a subset of AMLs may rely on increased LDL accumulation during treatment with particular drugs. Our data suggest that cholesterol synthesis inhibitors may improve the efficacy of standard antileukemia regimens, but that for maximum benefit, therapy may need to be tailored for individual patients with leukemia.

Description: Leukemic relapse after allogeneic hematopoietic cell transplantation (HCT) remains a major cause of treatment failure in patients (pts) who enter HCT with high-risk acute myeloid leukemia (AML). Therapeutic "graft-versus-leukemia" (GVL) effects are often accompanied by substantial morbidity and mortality caused by graft versus host disease (GVHD), as graft T cells have not been selected for specificity for leukemia antigens. To selectively promote GVL without inducing GVHD, we first developed a therapy employing CD8+ cytotoxic T lymphocyte (CTL) clones targeting Wilms' Tumor Antigen 1 (WT1), a non-polymorphic protein over-expressed 10-1000x by leukemic cells compared to normal CD34+ cells. Although CTL clones derived from HLA matched donors infused in the corresponding pts were safe, the anti-leukemic efficacy was limited in part due to a wide variability of functional avidities obtained for each patient-donor pair and limited persistence of CTL clones (Chapuis A. et al., STM 2013). We therefore identified and characterized a native, high affinity WT1-specific TCR (TCRC4), isolated from screening the peripheral repertoires of healthy HLA A*0201+ donors. Thus this TCR had been subjected to negative thymic selection, which should minimize the potential risk of off-target toxicity. To enhance persistence, we inserted TCRC4 in EBV- or CMV-specific donor substrate cells, with the former preferred based on the higher frequency of central memory (TCM). Twenty-two HLA A*0201+ pts received up to 1010 TCRC4 transduced donor-derived virus-specific cytotoxic T cells (CTL)/m2, following recovery of hematopoiesis after a matched HCT transplant for AML (NCT01640301). Adverse events included expected transient (3% beyond 7 days)(Figure 2). Analysis of donor virus-specific populations demonstrated that EBV- compared to CMV-specific cells expressed significantly higher long-lived memory and decreased exhaustion/activation markers, supporting results suggesting human cells derived from predominantly TCM populations are imprinted with a program that enhances post-transfer survival. CTLEBV/TCR-C4 that persisted at high frequencies in the PA not only exhibited an effector phenotype (i.e., did not convert to TCM as had been previously observed) but also expressed Ki67, suggesting continued activation. The transferred CTLEBV/TCR-C4 maintained the ability to secrete IFNg, TNFa and IL-2, preferentially through TCRC4 (Figure 3), and, as no ongoing EBV-reactivation was detected in serum, the results strongly suggest continued WT1-antigen encounter as the driver of proliferation/persistence and a contributing mechanism for AML relapse prevention in the PA. Furthermore, although the infused CTLEBV/TCR-C4 were polyclonal, the dominant clonotypes in the infusion product also composed the majority of persisting cells, suggesting rare "fit" clonotypes expand during cell production and lead to oligoclonal survival in vivo. Disclosures Chapuis: Juno Therapeutics Inc: Research Funding. Yeung:Gilead Sciences: Research Funding. Greenberg:Juno Therapeutics Inc: Equity Ownership, Research Funding.

Description: The mevalonate pathway produces many critical substances in cells, including sterols essential for membrane structure and isoprenoids vital to the function of many membrane proteins. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is a rate-limiting enzyme in the mevalonate pathway. Because cholesterol is a product of this pathway, HMG-CoA reductase inhibitors (statins) are used to treat hypercholesterolemia. Statins are also toxic to several malignancies, including acute myeloid leukemia (AML). Although this toxicity has been attributed to the inhibition of Ras/Rho isoprenylation, we have previously shown that statin toxicity in primary AML cells (AMLs) does not correlate with Ras isoprenylation or with activating Ras mutations. In other studies, we have shown that hypoxic and oxidant injuries induce cholesterol increments in renal tubule cells and that statins sensitize these cells to injury by blocking protective cholesterol responses. We now demonstrate that exposing particular AMLs to radiochemotherapy induces much greater cellular cholesterol increments than those seen in similarly treated normal bone marrow. Treatment of these AMLs with mevastatin or zaragozic acid (which inhibits cholesterol synthesis but not isoprenoid synthesis) attenuates the cholesterol increments and sensitizes cells to radiochemotherapy. The extent of toxicity is affected by the availability of extracellular lipoproteins, further suggesting that cellular cholesterol is critical to cell survival in particular AMLs. Because zaragozic acid does not inhibit isoprenoid synthesis, these data suggest that cholesterol modulation is an important mechanism whereby statins exert toxic effects on some AMLs and that cholesterol modulators may improve therapeutic ratios in AML by impacting cholesterol-dependent cytoresistance.