Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.

Flavopiridol is a small molecule inhibitor of cyclin-dependent kinases (CDK) known to impair global transcription via inactivation of positive transcription elongation factor b. It has been demonstrated to have significant activity predominantly in chronic lymphocytic leukemia and acute myeloid leukemia in phase I/II clinical trials while other similar CDK inhibitors are vigorously being pursued in pre-clinical and clinical studies. Although flavopiridol is a potent therapeutic agent against blood diseases, some patients still have primary or acquired resistance throughout their clinical course. Considering the limited knowledge of resistance mechanisms of flavopiridol, we investigated the potential mechanisms of resistance to flavopiridol in a cell line system, which gradually acquired resistance to flavopiridol in vitro, and then confirmed the mechanism in patient samples. Herein, we present that this resistant cell line developed resistance through up-regulation of phosphorylation of RNA polymerase II C-terminal domain, activation of CDK9 kinase activity, and prolonged Mcl-1 stability to counter flavopiridol's drug actions. Further analyses suggest MAPK/ERK activation-mediated Mcl-1 stabilization contributes to the resistance and knockdown of Mcl-1 in part restores sensitivity to flavopiridol-induced cytotoxicity. Altogether, these findings demonstrate that CDK9 is the most relevant target of flavopiridol and provide avenues to improve the therapeutic strategies in blood malignancies.

A growing body of evidence suggests that the resistance of CLL cells to apoptosis is partly mediated through the interactions between leukemia cells and adjacent stromal cells residing in the lymphatic tissue or bone marrow microenvironment. Mcl-1, an anti-apoptotic protein that is associated with failure to treatment is up-regulated in CLL lymphocytes after interaction with microenvironment. However, the regulation of its expression in context to microenvironment is unclear. We evaluated and compared changes in Mcl-1 in CLL B-cells in suspension culture and when co-cultured on stromal cells. The blockade of apoptosis in co-cultured CLL cells is associated with diminution in caspase-3 and PARP cleavage and is not dependent on cytogenetic profile or prognostic factors of the disease. Stroma-derived resistance to apoptosis is associated with a cascade of transcriptional events such as increase in levels of total RNA Pol II and its phosphorylation at Ser2 and Ser5, increase in the rate of global RNA synthesis, and amplification of Mcl-1 transcript levels. The latter is associated with increase in Mcl-1 protein level without an impact on the levels of Bcl-2 and Bcl-xL. Post-translational modifications of protein kinases show increased phosphorylation of Akt at Ser473, Erk at Thr202/Tyr204 and Gsk-3β at Ser9 and augmentation of total Mcl-1 accumulation along with phosphorylation at Ser159/Thr163 sites. Collectively, stroma-induced apoptosis resistance is mediated through signaling proteins that regulate transcriptional and translational expression and post-translational modification of Mcl-1 in CLL cells in context to bone marrow stromal microenvironment.

Ibrutinib compared with ofatumumab significantly improves progression-free and overall survival in patients with previously treated chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).

Results of RESONATE-2 (PCYC-1115/1116) supported approval of ibrutinib for first-line treatment of chronic lymphocytic leukemia. Extended analysis of RESONATE-2 was conducted to determine long-term efficacy and safety of ibrutinib in older patients with chronic lymphocytic leukemia. A total of 269 patients aged ≥65 years with previously untreated chronic lymphocytic leukemia without del(17p) were randomized 1:1 to ibrutinib (n=136) or chlorambucil (n=133) on days 1 and 15 of a 28-day cycle for 12 cycles. Median ibrutinib treatment duration was 28.5 months. Ibrutinib significantly prolonged progression-free survival versus chlorambucil (median, not reached vs 15 months; hazard ratio, 0.12; 95% confidence interval, 0.07-0.20; P<0.0001). The 24-month progression-free survival was 89% with ibrutinib (97% and 89% in patients with del[11q] and unmutated immunoglobulin heavy chain variable region gene, respectively). Progression-free survival rates at 24 months were also similar regardless of age (<75 years [88%], ≥75 years [89%]). Overall response rate was 92% (125/136). Rate of complete response increased substantially from 7% at 12 months to 18% with extended follow up. Greater quality of life improvements occurred with ibrutinib versus chlorambucil in Functional Assessment of Chronic Illness Therapy-Fatigue (P=0.0013). The most frequent grade ≥3 adverse events were neutropenia (12%), anemia (7%), and hypertension (5%). Rate of discontinuations due to adverse events was 12%. Results demonstrated that first-line ibrutinib for elderly patients with chronic lymphocytic leukemia provides sustained response and progression-free survival benefits over chemotherapy, with depth of response improving over time without new toxicity concerns. This trial was registered at clinicaltrials.gov identifier 01722487 and 01724346.

Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts (change >0.1 in clonal cancer cell fraction, Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones.

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton's tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism-associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell-mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre-germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD.

The constitutive activation of B-cell receptor (BCR) signaling, together with the overexpression of the Bcl-2 family anti-apoptotic proteins, represents two hallmarks of chronic lymphocytic leukemia (CLL) that drive leukemia cell proliferation and sustain their survival. TG02 is a small molecule multi-kinase inhibitor that simultaneously targets both of these facets of CLL pathogenesis. First, its inhibition of cyclin-dependent kinase 9 blocked the activation of RNA polymerase II and transcription. This led to the depletion of Mcl-1 and rapid induction of apoptosis in the primary CLL cells. This mechanism of apoptosis was independent of CLL prognostic factors or prior treatment history, but dependent on the expression of BAX and BAK. Second, TG02, which inhibits the members of the BCR signaling pathway such as Lck and Fyn, blocked BCR-crosslinking-induced activation of NF-κB and Akt, indicating abrogation of BCR signaling. Finally, the combination of TG02 and ibrutinib demonstrated moderate synergy, suggesting a future combination of TG02 with ibrutinib, or use in patients that are refractory to the BCR antagonists. Thus, the dual inhibitory activity on both the CLL survival pathway and BCR signaling identifies TG02 as a unique compound for clinical development in CLL and possibly other B cell malignancies.

Ibrutinib, a once-daily oral inhibitor of Bruton's tyrosine kinase, is approved in the United States and Europe for treatment of patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). The phase 3 RESONATE study showed improved efficacy of single-agent ibrutinib over ofatumumab in patients with relapsed/refractory CLL/SLL, including those with high-risk features. Here we report the final analysis from RESONATE with median follow-up on study of 65.3 months (range, 0.3-71.6) in the ibrutinib arm. Median progression-free survival (PFS) remained significantly longer for patients randomized to ibrutinib vs ofatumumab (44.1 vs 8.1 months; hazard ratio [HR]: 0.148; 95% confidence interval [CI]: 0.113-0.196; P˂.001). The PFS benefit with ibrutinib vs ofatumumab was preserved in the genomic high-risk population with del(17p), TP53 mutation, del(11q), and/or unmutated IGHV status (median PFS 44.1 vs 8.0 months; HR: 0.110; 95% CI: 0.080-0.152), which represented 82% of patients. Overall response rate with ibrutinib was 91% (complete response/complete response with incomplete bone marrow recovery, 11%). Overall survival, censored for crossover, was better with ibrutinib than ofatumumab (HR: 0.639; 95% CI: 0.418-0.975). With up to 71 months (median 41 months) of ibrutinib therapy, the safety profile remained consistent with prior reports; cumulatively, all-grade (grade ≥3) hypertension and atrial fibrillation occurred in 21% (9%) and 12% (6%) of patients, respectively. Only 16% discontinued ibrutinib because of adverse events (AEs). These long-term results confirm the robust efficacy of ibrutinib in relapsed/refractory CLL/SLL irrespective of high-risk clinical or genomic features, with no unexpected AEs. This trial is registered at www.clinicaltrials.gov (NCT01578707).

We developed a simple, rapid and quantitative assay using the fluorescent probe PicoGreen to measure the concentration of ionizing radiation-induced double-stranded DNA (dsDNA) in mouse plasma, and we correlated this concentration with the radiation dose. With 70 μl of blood obtained by fingerstick, this 30 min assay reduces protein interference without extending sample processing time. Plasma from nonirradiated mice (BALB/c and NIH Swiss) was pooled, diluted and spiked with dsDNA to establish sensitivity and reproducibility of the assay to quantify plasma dsDNA. The assay was then used to directly quantify dsDNA in plasma at 0-48 h after mice received 0-10 Gy total-body irradiation (TBI). There are three optimal conditions for this assay: 1:10 dilution of plasma in water; 1:200 dilution of PicoGreen reagent in water; and calibration of radiation-induced dsDNA concentration through a standard addition method using serial spiking of samples with genomic dsDNA. Using the internal standard calibration curve of the spiked samples method, the signal developed within 5 min, exhibiting a linear signal (r(2) = 0.997). The radiation-induced elevation of plasma DNA in mice started at 1-3 h, peaked at 9 h and gradually returned to baseline at 24 h after TBI (6 Gy). DNA levels in plasma collected from mice 9 h after 0-10 Gy TBI correlated strongly with dose (r(2) = 0.991 and 0.947 for BALB/c and NIH Swiss, respectively). Using the PicoGreen assay, we observed a radiation dose-dependent response in extracellular plasma DNA 9 h after irradiation with an assay time ≤ 30 min.

Tyrosine-kinase inhibitors improve overall survival in patients with chronic myeloid leukaemia in chronic phase (CML-CP). Survival compared with the general population by age, response, and type of tyrosine-kinase inhibitor is not known. With use of data from trials of tyrosine kinase inhibitors, we compared overall survival in patients with newly diagnosed CML-CP to that of general population.

Resistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.

The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Deltaexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing.

Adults with relapsed/refractory acute lymphoblastic leukemia (R/R ALL) have a poor prognosis, especially if disease burden is high. This post hoc analysis of the phase 3 INO-VATE trial examined the efficacy and safety of inotuzumab ozogamicin (InO) vs. standard of care chemotherapy (SC) among R/R ALL patients with low, moderate, or high disease burden, respectively, defined as bone marrow blasts (BMB) < 50% (n = 53 vs. 48), 50-90% (n = 79 vs. 83), and >90% (n = 30 vs. 30). Patients in the InO vs. SC arm with low, moderate, and high BMB%, respectively, had improved rates of complete remission/complete remission with incomplete hematologic recovery (74% vs. 46% [p = 0.0022], 75 vs. 27% [p < 0.0001], and 70 vs. 17% [p < 0.0001]), and improved overall survival (hazard ratio: 0.64 [p = 0.0260], 0.81 [p = 0.1109], and 0.60 [p = 0.0335]). Irrespective of BMB%, cytopenias were the most common treatment-emergent adverse events, and post-transplant veno-occlusive disease was more common with InO vs. SC. Patients with extramedullary disease or lymphoblastic lymphoma showed similar efficacy and safety outcomes. This favorable benefit-to-risk ratio of InO treatment irrespective of disease burden supports its use in challenging and high disease burden subpopulations. INO-VATE is registered at www.clinicaltrials.gov : #NCT01564784.

We report long-term follow-up from the RESONATE-2 phase 3 study of the once-daily Bruton's tyrosine kinase inhibitor ibrutinib, which is the only targeted therapy with significant progression-free survival (PFS) and overall survival (OS) benefit in multiple randomized chronic lymphocytic leukemia (CLL) studies. Patients (≥65 years) with previously untreated CLL, without del(17p), were randomly assigned 1:1 to once-daily ibrutinib 420 mg until disease progression/unacceptable toxicity (n = 136) or chlorambucil 0.5-0.8 mg/kg ≤12 cycles (n = 133). With up to 8 years of follow-up (range, 0.1-96.6 months; median, 82.7 months), significant PFS benefit was sustained for ibrutinib vs chlorambucil (hazard ratio [HR], 0.154; 95% confidence interval [CI], 0.108-0.220). At 7 years, PFS was 59% for ibrutinib vs 9% for chlorambucil. PFS benefit was also observed for ibrutinib- vs chlorambucil-randomized patients with high-risk genomic features: del(11q) (HR, 0.033; 95% CI, 0.010-0.107) or unmutated immunoglobulin heavy chain variable region (HR, 0.112; 95% CI, 0.065-0.192). OS at 7 years was 78% with ibrutinib. Prevalence of adverse events (AEs) was consistent with previous 5-year follow-up. Ibrutinib dosing was held (≥7 days) for 79 patients and reduced for 31 patients because of AEs; these AEs resolved or improved in 85% (67 of 79) and 90% (28 of 31) of patients, respectively. With up to 8 years of follow-up, 42% of patients remain on ibrutinib. Long-term RESONATE-2 data demonstrate sustained benefit with first-line ibrutinib treatment for CLL, including for patients with high-risk genomic features. These trials were registered at www.clinicaltrials.gov as #NCT01722487 and #NCT01724346.

ELEVATE-RR demonstrated noninferior progression-free survival and lower incidence of key adverse events (AEs) with acalabrutinib vs ibrutinib in previously treated chronic lymphocytic leukemia. We further characterize AEs of acalabrutinib and ibrutinib via post hoc analysis. Overall and exposure-adjusted incidence rate was assessed for common Bruton tyrosine kinase inhibitor-associated AEs and for selected events of clinical interest (ECIs). AE burden scores based on previously published methodology were calculated for AEs overall and selected ECIs. Safety analyses included 529 patients (acalabrutinib, n = 266; ibrutinib, n = 263). Among common AEs, incidences of any-grade diarrhea, arthralgia, urinary tract infection, back pain, muscle spasms, and dyspepsia were higher with ibrutinib, with 1.5- to 4.1-fold higher exposure-adjusted incidence rates. Incidences of headache and cough were higher with acalabrutinib, with 1.6- and 1.2-fold higher exposure-adjusted incidence rate, respectively. Among ECIs, incidences of any-grade atrial fibrillation/flutter, hypertension, and bleeding were higher with ibrutinib, as were exposure-adjusted incidence rates (2.0-, 2.8-, and 1.6-fold, respectively); incidences of cardiac events overall (the Medical Dictionary for Regulatory Activities system organ class) and infections were similar between arms. Rate of discontinuation because of AEs was lower for acalabrutinib (hazard ratio, 0.62; 95% confidence interval, 0.41-0.93). AE burden score was higher for ibrutinib vs acalabrutinib overall and for the ECIs atrial fibrillation/flutter, hypertension, and bleeding. A limitation of this analysis is its open-label study design, which may influence the reporting of more subjective AEs. Overall, event-based analyses and AE burden scores demonstrated higher AE burden overall and specifically for atrial fibrillation, hypertension, and hemorrhage with ibrutinib vs acalabrutinib. This trial was registered at www.clinicaltrials.gov as #NCT02477696.

We developed and validated a two-gene signature that predicts prognosis in previously-untreated chronic lymphocytic leukemia (CLL) patients. Using a 65 sample training set, from a cohort of 131 patients, we identified the best clinical models to predict time-to-treatment (TTT) and overall survival (OS). To identify individual genes or combinations in the training set with expression related to prognosis, we cross-validated univariate and multivariate models to predict TTT. We identified four gene sets (5, 6, 12, or 13 genes) to construct multivariate prognostic models. By optimizing each gene set on the training set, we constructed 11 models to predict the time from diagnosis to treatment. Each model also predicted OS and added value to the best clinical models. To determine which contributed the most value when added to clinical variables, we applied the Akaike Information Criterion. Two genes were consistently retained in the models with clinical variables: SKI (v-SKI avian sarcoma viral oncogene homolog) and SLAMF1 (signaling lymphocytic activation molecule family member 1; CD150). We optimized a two-gene model and validated it on an independent test set of 66 samples. This two-gene model predicted prognosis better on the test set than any of the known predictors, including ZAP70 and serum β2-microglobulin.

Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.

CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.