Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells.

BACKGROUND: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. AIMS AND METHODS: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion. RESULTS: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. CONCLUSIONS: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.

Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity.

Chimeric antigen receptors (CARs) significantly enhance the anti-tumor activity of immune effector cells. Although most studies have evaluated CAR expression in T cells, here we evaluate different CAR constructs that improve natural killer (NK) cell-mediated killing. We identified a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling. NK cells derived from human iPSCs that express this CAR (NK-CAR-iPSC-NK cells) have a typical NK cell phenotype and demonstrate improved anti-tumor activity compared with T-CAR-expressing iPSC-derived NK cells (T-CAR-iPSC-NK cells) and non-CAR-expressing cells. In an ovarian cancer xenograft model, NK-CAR-iPSC-NK cells significantly inhibited tumor growth and prolonged survival compared with PB-NK cells, iPSC-NK cells, or T-CAR-iPSC-NK cells. Additionally, NK-CAR-iPSC-NK cells demonstrate in vivo activity similar to that of T-CAR-expressing T cells, although with less toxicity. These NK-CAR-iPSC-NK cells now provide standardized, targeted "off-the-shelf" lymphocytes for anti-cancer immunotherapy.

Mouse Xenograft Model for Intraperitoneal Administration of NK Cell Immunotherapy for Ovarian Cancer.

Natural killer (NK) cells are an attractive cell population for immunotherapy. Adoptive transfer of NK cells has been tested in multiple clinical trials including acute myeloid leukemia (AML) and ovarian cancer, although limitations do exist especially for treatment of solid tumors. In order to overcome these limitations, mouse xenograft models are needed for evaluation of various NK cell populations, as well as routes of NK cell administration. Here, we describe the methods used for the establishment of an intraperitoneal (ip) ovarian cancer mouse xenograft model with ip delivery of NK cells. This model has been successfully employed with multiple ovarian cell lines and could be applied to other tumor models where the tumor's primary location is in the peritoneal cavity. It is also compatible with multiple routes of NK cell administration. Bioluminescent imaging for monitoring tumor formation and response provides for easy visualization of NK cell tumor inhibition. This xenograft model is superior to other models because the tumor is implanted into the same physiological space where ovarian cancer is found, which allows for improved mimicking of actual disease.

Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer.

Natural killer (NK) cells can provide effective immunotherapy for ovarian cancer. Here, we evaluated the ability of NK cells isolated from peripheral blood (PB) and NK cells derived from induced pluripotent stem cell (iPSC) to mediate killing of ovarian cancer cells in a mouse xenograft model. A mouse xenograft model was used to evaluate the intraperitoneal delivery of three different NK cell populations: iPSC-derived NK cells, PB-NK cells that had been activated and expanded in long-term culture, and overnight activated PB-NK cells that were isolated through CD3/CD19 depletion of PB B and T cells. Bioluminescent imaging was used to monitor tumor burden of luciferase expressing tumor lines. Tumors were allowed to establish prior to administering NK cells via intraperitoneal injection. These studies demonstrate a single dose of any of the three NK cell populations significantly reduced tumor burden. When mice were given three doses of either iPSC-NK cells or expanded PB-NK cells, the median survival improved from 73 days in mice untreated to 98 and 97 days for treated mice, respectively. From these studies, we conclude iPSC-derived NK cells mediate antiovarian cancer killing at least as well as PB-NK cells, making these cells a viable resource for immunotherapy for ovarian cancer. Due to their ability to be easily differentiated into NK cells and their long-term expansion potential, iPSCs can be used to produce large numbers of well-defined NK cells that can be banked and used to treat a large number of patients including treatment with multiple doses if necessary.

Utilizing chimeric antigen receptors to direct natural killer cell activity.

Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains.

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model.

BACKGROUND AIMS: There is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion. METHODS: An ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays. RESULTS: IP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion. CONCLUSIONS: IP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.

Overexpression of Mcl-1 confers multidrug resistance, whereas topoisomerase IIß downregulation introduces mitoxantrone-specific drug resistance in acute myeloid leukemia.

Drug resistance is a serious challenge in cancer treatment and can be acquired through multiple mechanisms. These molecular changes may introduce varied extents of resistance to different therapies and need to be characterized for optimal therapy choice. A recently discovered small molecule, ethyl-2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate) (CXL017), reveals selective cytotoxicity toward drug-resistant leukemia. A drug-resistant acute myeloid leukemia cell line, HL60/MX2, also failed to acquire resistance to CXL017 upon chronic exposure and regained sensitivity toward standard therapies. In this study, we investigated the mechanisms responsible for HL60/MX2 cells' drug resistance and the molecular basis for its resensitization. Results show that the HL60/MX2 cell line has an elevated level of Mcl-1 protein relative to the parental cell line, HL60, and its resensitized cell line, HL60/MX2/CXL017, whereas it has a reduced level of topoisomerase IIß. Mcl-1 overexpression in HL60/MX2 cells is mainly regulated through phospho-extracellular signal-regulated protein kinases 1 and 2-mediated Mcl-1 stabilization, whereas the reduction of topoisomerase IIß in HL60/MX2 cells is controlled through genetic downregulation. Upregulating Mcl-1 introduces multidrug resistance to standard therapies, whereas its downregulation results in significant cell death. Downregulating topoisomerase IIß confers resistance specifically to mitoxantrone, not to other topoisomerase II inhibitors. Overall, these data suggest that Mcl-1 overexpression is a critical determinant for cross-resistance to standard therapies, whereas topoisomerase IIß downregulation is specific to mitoxantrone resistance.

Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy.

Adoptive transfer of antitumor lymphocytes has gained intense interest in the field of cancer therapeutics over the past two decades. Human natural killer (NK) cells are a promising source of lymphocytes for anticancer immunotherapy. NK cells are part of the innate immune system and exhibit potent antitumor activity without need for human leukocyte antigen matching and without prior antigen exposure. Moreover, the derivation of NK cells from pluripotent stem cells could provide an unlimited source of lymphocytes for off-the-shelf therapy. To date, most studies on hematopoietic cell development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have used incompletely defined conditions and been on a limited scale. Here, we have used a two-stage culture system to efficiently produce NK cells from hESCs and iPSCs in the absence of cell sorting and without need for xenogeneic stromal cells. This novel combination of embryoid body formation using defined conditions and membrane-bound interleukin 21-expressing artificial antigen-presenting cells allows production of mature and functional NK cells from several different hESC and iPSC lines. Although different hESC and iPSC lines had varying efficiencies in hematopoietic development, all cell lines tested could produce functional NK cells. These methods can be used to generate enough cytotoxic NK cells to treat a single patient from fewer than 250,000 input hESCs/iPSCs. Additionally, this strategy provides a genetically amenable platform to study normal NK cell development and education in vitro.

Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017): a novel scaffold that resensitizes multidrug resistant leukemia cells to chemotherapy.

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer, and there is a pressing need for new therapies. We have recently developed ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017), derived from a dual inhibitor of Bcl-2 and SERCA proteins, sHA 14-1, with selective cytotoxicity toward MDR cancer cell lines in vitro. In this study, we present new evidence for its therapeutic potential in treatment of MDR cancers and offer mechanistic insights toward its preferential targeting of drug-resistant cancer. CXL017 selectively suppressed the growth of tumors derived from the MDR cancer cell line, HL60/MX2, in vivo. In addition, even after chronic exposure to CXL017, HL60/MX2 failed to develop stable resistance to CXL017, whereas it acquired >2000-fold resistance to cytarabine (Ara-C), the major first-line chemotherapy for the treatment of acute myeloid leukemia (AML). Remarkably, instead of acquiring further cross-resistance, HL60/MX2 cells exposed to CXL017 were resensitized to standard therapies (10- to 100-fold). Western blotting analyses revealed that CXL017 exposure significantly down-regulated Mcl-1 and Bax and up-regulated Noxa, Bim, Bcl-X(L), SERCA2, and SERCA3 proteins, along with a reduction in endoplasmic reticulum (ER) calcium content. Given the well-established functions of Bcl-2 family proteins and ER calcium in drug resistance, our results suggest that the down-regulation of Mcl-1 and the up-regulation of Noxa and Bim along with the decrease in ER calcium content are likely responsible for CXL017-induced resensitization of MDR cancer cells. These data also demonstrate the unique potential of CXL017 to overcome MDR in cancer treatment.

Structure-activity relationship (SAR) study of ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) and the potential of the lead against multidrug resistance in cancer treatment.

Multidrug resistance (MDR) against standard therapies poses a serious challenge in cancer treatment, and there is a clinical need for new anticancer agents that would selectively target MDR malignancies. Our previous studies have identified a 4H-chromene system, CXL017 (4) as an example, that can preferentially kill MDR cancer cells. To further improve its potency, we have performed detailed structure-activity relationship (SAR) studies at the 3, 4, and 6 positions of the 4H-chromene system. The results reveal that the 3 and 4 positions prefer rigid and hydrophobic functional groups while the 6 position prefers a meta or para-substituted aryl functional group and the substituent should be small and hydrophilic. We have also identified and characterized nine MDR cancer cells that acquire MDR through different mechanisms and demonstrated the scope of our new lead, 9g, to selectively target different MDR cancers, which holds promise to help manage MDR in cancer treatment.

Lung tumorigenesis suppressing effects of a commercial kava extract and its selected compounds in A/J mice.

Lung cancer is the most deadly malignancy in the US. Chemoprevention is potentially a complementary approach to smoking cessation for lung cancer control. Recently, we reported that a commercially available form of kava extract significantly inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo(a)pyrene (BaP)-induced lung tumorigenesis in A/J mice at a dose of 10 mg per gram diet. In the present study, we examined the dose-dependent lung tumor inhibitory activities of kava and investigated potential active constituent(s). Mice treated with carcinogen alone contained 12.1±5.8 lung adenomas per mouse 22 weeks after final carcinogen administration. Mice that were fed diets containing kava at dosages of 1.25, 2.5, 5, and 10 mg/g of diet had 8.4±3.5, 6.6±3.5, 4.3±2.4, and 3.8±2.3 lung adenomas per mouse, respectively. This corresponds to a reduction of 31%, 46%, 65% and 69% in tumor multiplicity, which were all statistically significant (p < 0.05). Analyses of lung adenoma tissues derived from kava-treated animals revealed that kava significantly inhibited adenoma cell proliferation while it had no detectable effect on cell death, indicating that kava primarily suppressed lung tumorigenesis in A/J mice via inhibition of cell proliferation. Flavokawains A, B, and C, three chalcone-based components from kava, demonstrated greatly reduced chemopreventive efficacies even at concentrations much higher than their natural abundance, suggesting that they alone were unlikely to be responsible for kava's chemopreventive activity. Kava at all dosages and treatment regimens did not induce detectable adverse effects, particularly with respect to liver. Specifically, kava treatment showed no effect on liver integrity indicator enzymes or liver weight, indicating that kava may be potentially safe for long-term chemopreventive application.

Structure-activity relationship and molecular mechanisms of ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) and its analogues.

Multidrug resistance (MDR) in cancer is a phenomenon in which administration of a single chemotherapeutic agent causes cross-resistance of cancer cells to a variety of therapies even with different mechanisms of action. Development of MDR against standard therapies is a major challenge in the treatment of cancer. Previously we have demonstrated a unique ability of CXL017 (5) to selectively target MDR cancer cells and synergize with mitoxantrone (MX) in HL60/MX2 MDR cells. Here we expand its scope and demonstrate that 5 can synergize with both vincristine and paclitaxel in three different MDR cell lines (HL60/DNR, K562/HHT300, and CCRF-CEM/VLB100). We also demonstrate that 5 has potent cytotoxicity in the NCI-60 panel of cell lines with an average IC(50) of 1.04 µM. In addition, 5 has a unique mechanism of action in comparison with standard agents in the NCI database based on COMPARE analysis. Further structure-activity relationship study led to the development of a more potent analogue, compound 7d, with an IC(50) of 640 nM in HL60/MX2. Additionally, one enantiomer of 5 is 13-fold more active than the less active enantiomer. Taken together, our study has led to the discovery of a series of analogues that selectively target drug-resistant cancer cells with the potential for the treatment of drug-resistant cancers.

Identification of methysticin as a potent and non-toxic NF-kappaB inhibitor from kava, potentially responsible for kava's chemopreventive activity.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays an essential role in cancer development. The results of our recent chemopreventive study demonstrate that kava, a beverage in the South Pacific Islands, suppresses NF-kappaB activation in lung adenoma tissues, potentially a mechanism responsible for kava's chemopreventive activity. Methysticin is identified as a potent NF-kappaB inhibitor in kava with minimum toxicity. Other kava constituents, including four kavalactones of similar structures to methysticin, demonstrate minimum activities in inhibiting NF-kappaB.

Structure-activity relationship and molecular mechanisms of ethyl 2-amino-4-(2-ethoxy-2-oxoethyl)-6-phenyl-4h-chromene-3-carboxylate (sha 14-1) and its analogues.

Rapid development of multiple drug resistance against current therapies is a major barrier in the treatment of cancer. Therefore, anticancer agents that can overcome acquired drug resistance in cancer cells are of great importance. Previously, we have demonstrated that ethyl 2-amino-4-(2-ethoxy-2-oxoethyl)-6-phenyl-4H-chromene-3-carboxylate (5a, sHA 14-1), a stable analogue of ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (6, HA 14-1), mitigates drug resistance and synergizes with a variety of cancer therapies in leukemia cells. Structure-activity relationship (SAR) studies of 5a guided the development of ethyl 2-amino-6-(3',5'-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (5q, CXL017), a compound with low micromolar cytotoxicity against a wide-range of hematologic and solid tumor cells. More excitingly, our studies of 5q in camptothecin (CCRF-CEM/C2) and mitoxantrone (HL-60/MX2) resistant cancer cells highlight its ability to selectively kill drug-resistant cells over parent cancer cells. 5q inhibits tumor cell growth through the induction of apoptosis, with detailed mechanism of its selectivity toward drug-resistant cancer cells under investigation. These results suggest that 5q is a promising candidate for treatment of cancers with multiple drug resistance.

Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria.

HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.