Many cancers show primary or acquired drug resistance due to the overexpression of efflux pumps. A novel mechanism to circumvent this is to integrate drugs, such as anthracycline antibiotics, with nanoparticle delivery vehicles that can bypass intrinsic tumor drug-resistance mechanisms. DNA nanoparticles serve as an efficient binding platform for intercalating drugs (e.g. anthracyclines doxorubicin and daunorubicin, which are widely used to treat acute leukemias) and enable precise structure design and chemical modifications, for example for incorporating targeting capabilities. Here, we utilize DNA nanostructures to circumvent daunorubicin drug resistance at clinically relevant doses in a leukemia cell line model. We report the fabrication of a rod-like DNA origami drug carrier that can be controllably loaded with daunorubicin. We further directly verify that nanostructure-mediated daunorubicin delivery leads to increased drug entry and retention in cells relative to free daunorubicin at equal concentrations, which yields significantly enhanced drug efficacy. Our results indicate that DNA origami nanostructures can circumvent efflux pump-mediated drug resistance in leukemia cells at clinically relevant drug concentrations and provide a robust DNA nanostructure design that could be implemented in a wide range of cellular applications due to its remarkably fast self-assembly (~5 minutes) and excellent stability in cell culture conditions.

Natural killer (NK)-cell count is predictive of chronic lymphoid leukemia (CLL) disease progression and their dysfunction is well documented, but the etiology of this is currently lacking. CLL cells have been shown to over-express HLA-E, the natural ligand for NKG2A expressed on NK-cells that generates a distinct negative signal relative to direct NK-cell cytotoxicity in other disease models. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mAb), monalizumab, we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in CLL patients. Our work confirmed overexpression of HLA-E on CLL B-cells and demonstrated NKG2A expression on CD56C/16C NK-cells from CLL patients. We also demonstrate that blocking NKG2A on CLL NK-cells was sufficient to restore direct cytotoxicity ability of NK-cells against HLA-E-expressing targets without impacting NK-cell mediated antibody-dependent cellular cytotoxicity. Additionally, we proved the specificity of monalizumab in blocking NKG2A through Fc-blocking mechanisms. This paper provides justification for the potential clinical utility of monalizumab in the treatment of patients with CLL.

The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib has transformed chronic lymphocytic leukemia (CLL) therapy but requires continuous administration. These factors have spurred interest in combination treatments. Unlike with chemotherapy, CD20-directed antibody therapy has not improved the outcome of BTKi treatment. Whereas CD20 antigen density on CLL cells decreases during ibrutinib treatment, the B-cell activating factor (BAFF) and its receptor (BAFF-R) remain elevated. Furthermore, BAFF signaling via noncanonical NF-κB remains elevated with BTKi treatment. Blocking BAFF interaction with BAFF-R by using VAY-736, a humanized defucosylated engineered antibody directed against BAFF-R, antagonized BAFF-mediated apoptosis protection and signaling at the population and single-cell levels in CLL cells. Furthermore, VAY-736 showed superior antibody-dependent cellular cytotoxicity compared with CD20- and CD52-directed antibodies used in CLL. VAY-736 exhibited in vivo activity as a monotherapy and, when combined with ibrutinib, produced prolonged survival compared with either therapy alone. The in vivo activity of VAY-736 is dependent upon immunoreceptor tyrosine–based activation motif (ITAM)–mediated activation of effector cells as shown by using an ITAM-deficient mouse model. Collectively, our findings support targeting the BAFF signaling pathway with VAY-736 to more effectively treat CLL as a single agent and in combination with ibrutinib.

As checkpoint inhibitor immunotherapies gain traction among cancer researchers and clinicians, the need grows for assays that can definitively phenotype patient immune cells. Herein, we present an 8-color flow cytometry panel for lineage and immune checkpoint markers and validate it using healthy human donor peripheral blood mononuclear cells (PBMCs). Flow cytometry data was generated on a BD LSR Fortessa and supported by Luminex multiplex soluble immunoassay. Our data showed significant variation between donors at both baseline and different stages of activation, as well as a trend in increasing expression of checkpoint markers on stimulated CD4 + and CD8 + T-cells with time. Soluble immune checkpoint quantification assays revealed that LAG-3, TIM-3, CTLA-4, and PD-1 soluble isoforms are upregulated after stimulation. This 8-color flow cytometry panel, supported here by soluble immunoassay, can be used to identify and evaluate immune checkpoints on T-lymphocytes in cryopreserved human PBMC samples. This panel is ideal for characterizing checkpoint expression in clinical samples for which cryopreservation is necessary.

Resistance to PI3K inhibitors including idelalisib remains poorly understood. The present study was designed to identify mechanisms underlying PI3K inhibitor resistance in CLL through WES, RNAseq and biochemical confirmation. We performed WES on matched tumor and germline samples from 28 relapsed CLL patients treated with PI3K inhibitors (idelalisib, 79% (22/28), pilaralisib 10.7% (3/28), voxtalisib 10.7% (3/28)). The median number of prior therapies was 4.5 and the adverse cytogenetic aberrations del(17p) and del(11q) were seen in 21% and 32% respectively. A median of two longitudinal tumor samples were sequenced per patient (Nsamples= 68). An average of 23 ± 12 (range = 4-58) non-silent somatic mutations were identified in the initial sample. The non-silent mutation rate was 0.64 ± 0.32 per MB (range = 0.11-1.61) and typical of CLL. Significantly mutated genes (based on MutSig2CV) included SF3B1 36%, TP53 21%, DDX3X 14% and BRAF 11%. The cohort was sub-divided into responders (R) (Ncases= 18, Nsamples= 47) and non-responders (NR) (Ncases= 10, Nsamples= 21). Among the 10 NR, 7 had been treated with idelalisib and 3 with voxtalisib. Comparison of the mutational landscape between R and NR showed that, notably, mutations in BRAF(N=5), MAP2K1(N=2), KRAS(N=2),XPO1(N=1), PLEKHA1(N=1), INPPL1(N=1) and NXF1(N=1) were exclusively found in NR. Moreover, MAPK pathway genes (KRAS, BRAF and MAP2K1) were the only recurrent somatic mutations present exclusively in NR. We inferred the cancer cell fraction (ccf) for each somatic mutation using ABSOLUTE and found that the ccf values for MAP2K1 (p=0.01), XPO1 and PLEKHA1 mutations increased with time, indicating a positive selection pressure. Phylogic analysis of 6 R and 4 NR with at least 3 serial samples identified a median of 2 dynamic subclones (change of at least 10% between any two timepoints) and showed enrichment of the "Reactome_RAF_MAP_Kinase_Cascade" pathway in NR (p=3x10-4, q=0.04). In addition, Gene Set Enrichment Analysis comparing RNAseq samples drawn prior to or after PI3K therapy in five of the patients from our cohort (1 primary NR and 4 progressors after response) demonstrated activation of the "Reactome_Prolonged_ERK_Activation_Events" pathway at the later time-points (p=2.3x10-4, q= 0.03). Given these findings, we further investigated the role of the ERK/MAPK pathway in idelalisib resistance. Western blot analysis of ERK1/2 phosphorylation in ex vivo idelalisib treated PBMCs from an initially responding patient who acquired resistance later showed that idelalisib failed to inhibit ERK1/2 phosphorylation only at the resistant timepoint. This persistent ERK1/2 activation was inhibited by combination treatment with the MAP2K1 inhibitor CI-1040. We then profiled pAKT and pERK relative to total, by western blot on ex vivo idelalisib-treated samples (5 R, 5 NR). Idelalisib treatment ex vivo inhibited AKT phosphorylation in both R and NR, but no reduction in ERK phosphorylation was seen in NR, in contrast to R. Based on these findings we hypothesized that increased or persistent ERK activity confers resistance to PI3K inhibitors and tested the impact of MAP2K1 mutations on ERK activity. Among NR, we identified two different mutations in MAP2K1, E203K and Q56P (2/10 NR vs 0/18 R), both previously described as activating in solid tumors. In a separate cohort of multiply relapsed CLL patients, we found 4 cases of MAP2K1 F53L mutations, also previously reported in solid tumors. We studied the functional significance of these MAP2K1 mutations by generating stable MEC1 CLL cell lines expressing HA tagged E203K, F53L or Q56P MAP2K1 mutants. We found that all mutant cell lines had elevated basal ERK1/2 phosphorylation (E203K p<0.001; F53L p<0.001; Q56P p<0.001). Moreover, idelalisib did not inhibit IgM-induced ERK1/2 phosphorylation in the mutants when compared to the controls expressing wild type MAP2K1 (E203K p<0.001; F53L p<0.001; Q56P p<0.001). Preliminary results using the ERK1/2 inhibitor SCH772984 showed that the combination of SCH772984 along with idelalisib effectively decreased ERK1/2 phosphorylation in the mutant cell lines compared to idelalisib alone. Taken together, these data implicate the ERK pathway in idelalisib resistance and suggest that ERK1/2 inhibitors either combined with idelalisib at therapy start or added to idelalisib at early progression might sensitize patients to PI3K delta therapy. Disclosures Brown: Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Invectys: Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; TG Therapeutics: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Gilead: Consultancy, Research Funding; Celgene: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy; Verastem: Consultancy, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Sun Pharmaceutical Industries: Research Funding; Loxo: Consultancy; Boehringer: Consultancy.