Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy.

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

Elucidation of Focal Adhesion Kinase as a Modulator of Migration and Invasion and as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia.

The retention and re-migration of Chronic Lymphocytic Leukemia cells into cytoprotective and proliferative lymphoid niches is thought to contribute to the development of resistance, leading to subsequent disease relapse. The aim of this study was to elucidate the molecular processes that govern CLL cell migration to elicit a more complete inhibition of tumor cell migration. We compared the phenotypic and transcriptional changes induced in CLL cells using two distinct models designed to recapitulate the peripheral circulation, CLL cell migration across an endothelial barrier, and the lymph node interaction between CLL cells and activated T cells. Initially, CLL cells were co-cultured with CD40L-expressing fibroblasts and exhibited an activated B-cell phenotype, and their transcriptional signatures demonstrated the upregulation of pro-survival and anti-apoptotic genes and overrepresentation of the NF-κB signaling pathway. Using our dynamic circulating model, we were able to study the transcriptomics and miRNomics associated with CLL migration. More than 3000 genes were altered when CLL cells underwent transendothelial migration, with an overrepresentation of adhesion and cell migration gene sets. From this analysis, an upregulation of the FAK signaling pathway was observed. Importantly, PTK2 (FAK) gene expression was significantly upregulated in migrating CLL cells (PTK2 Fold-change = 4.9). Here we demonstrate that TLR9 agonism increased levels of p-FAK (p ≤ 0.05), which could be prevented by pharmacological inhibition of FAK with defactinib (p ≤ 0.01). Furthermore, a reduction in CLL cell migration and invasion was observed when FAK was inhibited (p ≤ 0.0001), supporting a role for FAK in both CLL migration and tissue invasion. When taken together, our data highlights the potential for combining FAK inhibition with current targeted therapies as a more effective treatment regime for CLL.

Targeting the Non-Canonical NF-κB Pathway in Chronic Lymphocytic Leukemia and Multiple Myeloma.

In this study, we evaluated an NF-κB inducing kinase (NIK) inhibitor, CW15337, in primary chronic lymphocytic leukemia (CLL) cells, CLL and multiple myeloma (MM) cell lines and normal B- and T-lymphocytes. Basal NF-κB subunit activity was characterized using an enzyme linked immunosorbent assay (ELISA), and the effects of NIK inhibition were then assessed in terms of cytotoxicity and the expression of nuclear NF-κB subunits following monoculture and co-culture with CD40L-expressing fibroblasts, as a model of the lymphoid niche. CW15337 induced a dose-dependent increase in apoptosis, and nuclear expression of the non-canonical NF-κB subunit, p52, was correlated with sensitivity to CW15337 (p = 0.01; r2 = 0.39). Co-culture on CD40L-expressing cells induced both canonical and non-canonical subunit expression in nuclear extracts, which promoted in vitro resistance against fludarabine and ABT-199 (venetoclax) but not CW15337. Furthermore, the combination of CW15337 with fludarabine or ABT-199 showed cytotoxic synergy. Mechanistically, CW15337 caused the selective inhibition of non-canonical NF-κB subunits and the transcriptional repression of BCL2L1, BCL2A1 and MCL1 gene transcription. Taken together, these data suggest that the NIK inhibitor, CW15337, exerts its effects via suppression of the non-canonical NF-κB signaling pathway, which reverses BCL2 family-mediated resistance in the context of CD40L stimulation.

Proteomics-based identification of cancer-associated proteins in chronic lymphocytic leukaemia

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a neoplasm of B-cells characterized by variable prognosis. Exploring the proteome of CLL cells may provide insights into the disease. Therefore, eleven proteomics experiments were conducted on eleven primary CLL samples. RESULTS: We reported a CLL proteome consisting of 919 proteins (false discovery rate (FDR) 1%) whose identification was based on the sequencing of two or more distinct peptides (FDR of peptide sequencing 1%). Mass spectrometry-based protein identification was validated for four different proteins using Western blotting and specific antibodies in different CLL samples. Small sizes of nucleolin (~57 kDa and ~68 kDa) showed a potential association with good prognosis CLL cells (n = 8, p < 0.01). Compared with normal B-cells, CLL cells over-expressed thyroid hormone receptor-associated protein 3 (THRAP3; n = 9; p = 0.00007), which is implicated in cell proliferation; and heterochromatin protein 1-binding protein 3 (HP1BP3; n = 10; p = 0.0002), which promotes cell survival and tumourogenesis. A smaller form of HP1BP3, which may correspond to HP1BP3 isoform-2, was specifically identified in normal B-cells (n = 10; p = 0.0001). HP1BP3 and THRAP3 predicted poor prognosis of CLL (p 0.05). Consistently, THRAP3 and HP1BP3 were found to be associated with cancer-related pathways (p 0.05). CONCLUSIONS: Our findings add to the known proteome of CLL and confirm the prognostic importance of two novel cancer-associated proteins in this disease.

A retrospective observational study to evaluate the clinical outcomes and routine management of patients with chronic lymphocytic leukaemia treated with idelalisib and rituximab in the UK and Ireland (RETRO-idel).

Idelalisib (IDL) is an oral first-in-class phosphatidylinositol 3-kinase delta (PI3Kδ) inhibitor approved for chronic lymphocytic leukaemia (CLL) alongside rituximab (R) since 2014. However, little data exist on routine practice. The RETRO-idel was a protocol-led, retrospective study of 110 patients [n = 27 front-line (1L)] who received IDL-R. The primary end-point was clinical overall response rate (ORR). The median (range) follow-up of the whole cohort was 30·2 (0·1-51·9) months. The median (range) age was 72 (48-89) years. Tumour protein p53-disruption was common [100% 1L, 32·5% relapsed/refractory (R/R)]. The best ORR (intention-to-treat) was 88·2% (1L 96·3%, R/R 85·5%). Overall, the median event-free survival (mEFS) was 20·3 months and time-to-next treatment was 29·2 months. The mEFS for 1L patients was 18·7 months and R/R patients was 21·7 months. The 3-year overall survival was 56·1% (95% confidence interval 45·7-65·3). IDL was discontinued in 87·3% (n = 96). More patients discontinued due to adverse events in the front-line setting (1L 63·0% vs. R/R 44·6%) and due to progressive disease in R/R patients (20·5% vs. 3·7% in 1L). Lower respiratory tract infection/pneumonia were reported in 34·5% (Grade ≥3, 19·1%), diarrhoea in 30·9% (Grade ≥3, 6·4%), and colitis in 9·1% (Grade ≥3, 5·5%). Overall, these data describe clear efficacy for IDL-R in routine practice. No new safety signals were identified, although careful management of known toxicities is required.

Genome-wide association study identifies risk loci for progressive chronic lymphocytic leukemia.

Prognostication in patients with chronic lymphocytic leukemia (CLL) is challenging due to heterogeneity in clinical course. We hypothesize that constitutional genetic variation affects disease progression and could aid prognostication. Pooling data from seven studies incorporating 842 cases identifies two genomic locations associated with time from diagnosis to treatment, including 10q26.13 (rs736456, hazard ratio (HR) = 1.78, 95% confidence interval (CI) = 1.47-2.15; P = 2.71 × 10-9) and 6p (rs3778076, HR = 1.99, 95% CI = 1.55-2.55; P = 5.08 × 10-8), which are particularly powerful prognostic markers in patients with early stage CLL otherwise characterized by low-risk features. Expression quantitative trait loci analysis identifies putative functional genes implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. In this work we identify rs736456 and rs3778076 as prognostic in CLL, demonstrating that disease progression is determined by constitutional genetic variation as well as known somatic drivers.

TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide-DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, ß2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

Dexamethasone in Hospitalized Patients with Covid-19.

BACKGROUND: Coronavirus disease 2019 (Covid-19) is associated with diffuse lung damage. Glucocorticoids may modulate inflammation-mediated lung injury and thereby reduce progression to respiratory failure and death. METHODS: In this controlled, open-label trial comparing a range of possible treatments in patients who were hospitalized with Covid-19, we randomly assigned patients to receive oral or intravenous dexamethasone (at a dose of 6 mg once daily) for up to 10 days or to receive usual care alone. The primary outcome was 28-day mortality. Here, we report the final results of this assessment. RESULTS: A total of 2104 patients were assigned to receive dexamethasone and 4321 to receive usual care. Overall, 482 patients (22.9%) in the dexamethasone group and 1110 patients (25.7%) in the usual care group died within 28 days after randomization (age-adjusted rate ratio, 0.83; 95% confidence interval [CI], 0.75 to 0.93; P<0.001). The proportional and absolute between-group differences in mortality varied considerably according to the level of respiratory support that the patients were receiving at the time of randomization. In the dexamethasone group, the incidence of death was lower than that in the usual care group among patients receiving invasive mechanical ventilation (29.3% vs. 41.4%; rate ratio, 0.64; 95% CI, 0.51 to 0.81) and among those receiving oxygen without invasive mechanical ventilation (23.3% vs. 26.2%; rate ratio, 0.82; 95% CI, 0.72 to 0.94) but not among those who were receiving no respiratory support at randomization (17.8% vs. 14.0%; rate ratio, 1.19; 95% CI, 0.92 to 1.55). CONCLUSIONS: In patients hospitalized with Covid-19, the use of dexamethasone resulted in lower 28-day mortality among those who were receiving either invasive mechanical ventilation or oxygen alone at randomization but not among those receiving no respiratory support. (Funded by the Medical Research Council and National Institute for Health Research and others; RECOVERY ClinicalTrials.gov number, NCT04381936; ISRCTN number, 50189673.).

Novel pyrrolobenzodiazepine benzofused hybrid molecules inhibit NF-κB activity and synergise with bortezomib and ibrutinib in hematological cancers.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are incurable hematological malignancies that are pathologically linked with aberrant NF-κB activation. In this study, we identified a group of novel C8-linked benzofused Pyrrolo[2,1-c][1,4]benzodiazepines (PBD) monomeric hybrids capable of sequence-selective inhibition of NF-κB with low nanomolar LD50 values in CLL (n=46) and MM cell lines (n=5). The lead compound, DC-1-192, significantly inhibited NF-κB DNA binding after just 4h exposure and demonstrating inhibitory effects on both canonical and non-canonical NF-κB subunits. In primary CLL cells, sensitivity to DC-1-192 was inversely correlated with RelA subunit expression (r2=0.2) and samples with BIRC3 or NOTCH1 mutations showed increased sensitivity (P=0.001). RNA-sequencing and gene set enrichment analysis confirmed the over-representation of NF-κB regulated genes in the down-regulated gene list. Furthermore, In vivo efficacy studies in NOD/SCID mice, using a systemic RPMI 8226 human multiple myeloma xenograft model, showed that DC-1-192 significantly prolonged survival (P=0.017). In addition, DC1-192 showed synergy with bortezomib and ibrutinib; synergy with ibrutinib was enhanced when CLL cells were co-cultured on CD40L-expressing fibroblasts in order to mimic the cytoprotective lymph node microenvironment (P = 0.01). Given that NF-κB plays a role in both bortezomib and ibrutinib resistance mechanisms, these data provide a strong rationale for the use of DC-1-192 in the treatment of NF-κB-driven cancers, particularly in the context of relapsed/refractory disease.

Development of a high-throughput SARS-CoV-2 antibody testing pathway using dried blood spot specimens.

BACKGROUND: Serological assays for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have roles in seroepidemiology, convalescent plasma-testing, antibody durability and vaccine studies. Currently, SARS-CoV-2 serology is performed using serum/plasma collected by venepuncture. Dried blood spot (DBS) testing offers significant advantages as it is minimally invasive, avoids venepuncture with specimens being mailed to the laboratory. METHODS: A pathway utilizing a newborn screening laboratory infrastructure was developed using an enzyme-linked immunosorbent assay to detect IgG antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein in DBS specimens. Paired plasma and DBS specimens from SARS-CoV-2 antibody-positive and -negative subjects and polymerase chain reaction positive subjects were tested. DBS specimen stability, effect of blood volume and punch location were also evaluated. RESULTS: DBS specimens from antibody-negative (n = 85) and -positive (n = 35) subjects and polymerase chain reaction positive subjects (n = 11) had a mean (SD; range) optical density (OD) of 0.14 (0.046; 0.03-0.27), 0.98 (0.41; 0.31-1.64) and 1.12 (0.37; 0.49-1.54), respectively. An action value OD >0.28 correctly assigned all cases. The weighted Deming regression for comparison of the DBS and the plasma assay yielded: y = 0.004041 + 1.005x, r = 0.991, Sy/x 0.171, n = 82. Extraction efficiency of antibodies from DBS specimens was >99%. DBS specimens were stable for at least 28 days at ambient room temperature and humidity. CONCLUSIONS: SARS-CoV-2 IgG receptor-binding domain antibodies can be reliably detected in DBS specimens. DBS serological testing offers lower costs than either point of care or serum/plasma assays that require patient travel, phlebotomy and hospital/clinic resources; the development of a DBS assay may be particularly important for resource poor settings.

Potential Role of Oral Rinses Targeting the Viral Lipid Envelope in SARS-CoV-2 Infection.

Emerging studies increasingly demonstrate the importance of the throat and salivary glands as sites of virus replication and transmission in early COVID-19 disease. SARS-CoV-2 is an enveloped virus, characterized by an outer lipid membrane derived from the host cell from which it buds. While it is highly sensitive to agents that disrupt lipid biomembranes, there has been no discussion about the potential role of oral rinsing in preventing transmission. Here, we review known mechanisms of viral lipid membrane disruption by widely available dental mouthwash components that include ethanol, chlorhexidine, cetylpyridinium chloride, hydrogen peroxide, and povidone-iodine. We also assess existing formulations for their potential ability to disrupt the SARS-CoV-2 lipid envelope, based on their concentrations of these agents, and conclude that several deserve clinical evaluation. We highlight that already published research on other enveloped viruses, including coronaviruses, directly supports the idea that oral rinsing should be considered as a potential way to reduce transmission of SARS-CoV-2. Research to test this could include evaluating existing or specifically tailored new formulations in well-designed viral inactivation assays, then in clinical trials. Population-based interventions could be undertaken with available mouthwashes, with active monitoring of outcome to determine efficacy. This is an under-researched area of major clinical need.

Phase Ib Study of Tirabrutinib in Combination with Idelalisib or Entospletinib in Previously Treated Chronic Lymphocytic Leukemia.

PURPOSE: Bruton tyrosine kinase (BTK) inhibition alone leads to incomplete responses in chronic lymphocytic leukemia (CLL). Combination therapy may reduce activation of escape pathways and deepen responses. This open-label, phase Ib, sequential dose-escalation and dose-expansion study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of the selective BTK inhibitor tirabrutinib alone, in combination with the PI3K delta (PI3Kδ) inhibitor idelalisib, or with the spleen tyrosine kinase (SYK) inhibitor entospletinib in patients with relapsed/refractory CLL. PATIENTS AND METHODS: Patients received either tirabrutinib monotherapy (80 mg every day) or tirabrutinib 20-150 mg every day in combination with either idelalisib (50 mg twice a day or 100 mg every day) or entospletinib (200 mg or 400 mg every day). RESULTS: Fifty-three patients were included. Systemic tirabrutinib exposure was comparable between monotherapy and combination therapy. No MTD was identified. Across all treatment groups, the most common adverse event was diarrhea (43%, 1 patient grade ≥3); discontinuation due to adverse events was uncommon (13%). Objective response rates were 83%, 93%, and 100%, and complete responses were 7%, 7%, and 10% in patients receiving tirabrutinib, tirabrutinib/idelalisib, and tirabrutinib/entospletinib, respectively. As of February 21, 2019, 46 of 53 patients continue to receive treatment on study. CONCLUSIONS: Tirabrutinib in combination with idelalisib or entospletinib was well tolerated in patients with CLL, establishing an acceptable safety profile for concurrent selective inhibition of BTK with either PI3Kδ or SYK. This small study did not establish a superior efficacy of the combinations over tirabrutinib alone. This trial is registered at www.clinicaltrials.gov (NCT02457598).

Clinical utility of telomere length measurements in cancer.

Cancer remains one of the leading causes of death in the developed world and despite impressive advances in therapeutic modalities, only a small subset of patients are currently cured. The underlying genetic heterogeneity of cancers clearly plays a crucial role in determining both the clinical course of individual pathologies and their responses to standard treatments. Although every tumour is to some extent distinct, there are recurrent features of cancers that can be exploited as therapeutic targets and as prognostic and predictive biomarkers; one such attribute is telomere length. Here we discuss the utility of telomere length evaluation in cancer and describe some of the promise and challenges of bringing this into clinical practice.