Drug In Chronic Lymphocytic Leukemia Research Articles

The Novel Anti-Cancer Agent, SpiD3, Is Cytotoxic in CLL Cells Resistant to Ibrutinib or Venetoclax

Background: B-cell receptor (BCR) signaling is a central driver in chronic lymphocytic leukemia (CLL), along with the activation of pro-survival pathways (e.g., NF-κB) and aberrant anti-apoptotic mechanisms (e.g., BCL2) culminating to CLL cell survival and drug resistance. Front-line targeted therapies such as ibrutinib (BTK inhibitor) and venetoclax (BCL2 inhibitor) have radically improved CLL management. Yet, persisting CLL cells lead to relapse in ~20% of patients, signifying the unmet need of inhibitor-resistant refractory CLL. SpiD3 is a novel spirocyclic dimer of analog 19 that displays NF-κB inhibitory activity and preclinical anti-cancer properties. Recently, we have shown that SpiD3 inhibits CLL cell proliferation and induces cytotoxicity by promoting futile activation of the unfolded protein response (UPR) pathway and generation of reactive oxygen species (ROS), resulting in the inhibition of protein synthesis in CLL cells. Methods: We performed RNA-sequencing using CLL cells rendered resistant to ibrutinib and venetoclax to explore potential vulnerabilities in inhibitor-resistant and SpiD3-treated CLL cells. Results: The transcriptomic analysis of ibrutinib- or venetoclax-resistant CLL cell lines revealed ferroptosis, UPR signaling, and oxidative stress to be among the top pathways modulated by SpiD3 treatment. By examining SpiD3-induced protein aggregation, ROS production, and ferroptosis in inhibitor-resistant CLL cells, our findings demonstrate cytotoxicity following SpiD3 treatment in cell lines resistant to current front-line CLL therapeutics. Conclusions: Our results substantiate the development of SpiD3 as a novel therapeutic agent for relapsed/refractory CLL disease.

Treatment patterns in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma post covalent Bruton tyrosine kinase inhibitor treatment: a Japanese claims database study.

Standard treatment has not been established for patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after discontinuation of covalent Bruton tyrosine kinase inhibitor (cBTKi) therapy. This retrospective, administrative database (Medical Data Vision) study described the patient characteristics, treatment patterns, and factors associated with receiving post-first-cBTKi treatment in Japanese patients with CLL/SLL. Patients aged ≥18 years with confirmed CLL/SLL diagnosis and treated with anti-neoplastic drugs indicated for CLL/SLL between March 2013 and February 2022 were included. Patient characteristics at baseline (first line), first cBTKi exposure (first-cBTKi), post-first-cBTKi treatment received, and the treatment sequence of CLL drugs received first line through third line, were described. Time-to-event analyses used the Kaplan-Meier method. Multivariable logistic regression analysis was used to explore factors associated with receiving post-first-cBTKi treatment among patients who discontinued first-cBTKi treatment. Among 2,424 eligible patients (median age: 72.0 years, 61.9% male), 450 (18.6%) received cBTKi in any treatment line. Among patients treated with cBTKi, 273 (60.7%) discontinued treatment; 56.0% of them (n = 153/273) received subsequent treatment. Median duration of post-first-cBTKi treatment was 2.2 months (95% confidence interval [CI]: 1.8, 3.5). The most common regimens post-first-cBTKi were cBTKi therapy (47.7%), bendamustine-based therapy (17.0%), and venetoclax-based therapy (13.1%). Patients aged <75 years (odds ratio [OR] [95% CI]: 2.0 [1.2, 3.4]) and those who did not receive blood transfusion during cBTKi treatment (OR [95% CI]: 2.3 [1.3, 4.1]) were more likely to receive post-first-cBTKi treatment. In conclusion, Japanese patients with CLL/SLL received various treatments for short duration after first-cBTKi discontinuation.

TRAF3-Inactivated Chronic Lymphocytic Leukemia Cells Show an Enhanced Metabolic Plasticity That Can be Attenuated By Glutaminolysis and Mitochondrial Pyruvate Import Inhibition

I ntroduction: TRAF3 gene is located in 14q32.32 and has been identified as a tumor suppressor gene involved in B-cell survival, immune response, and cellular metabolism. TRAF3 is frequently encompassed within 14q deletion and affected by inactivating mutations in 4% of chronic lymphocytic leukemia (CLL) patients. Recently, our group found that CLL patients harboring biallelic TRAF3 inactivation (deletion and mutation) showed a dismal prognosis, with a short time to first treatment (Pérez Carretero, AJH 2022). However, the biological role of TRAF3 alterations in CLL pathogenesis remains to be elucidated. Thus, the aims of this study wereto assess the functional consequences of these alterations and to determine their impact on therapy response. Methods: Previously reported TRAF3 mutations were mimicked in a CRISPR/Cas9-edited CLL model by using the PGA1 cell line (PGA1- TRAF3 mut). Metabolomic (liquid chromatography-mass spectrometry), transcriptomic (RNAseq) and functional analyses were carried out. To evaluate the metabolic specificities of PGA1- TRAF3 mut cells, oxidative phosphorylation (OXPHOS) and glycolytic capacity were studied by assessing the mitochondrial oxygen consumption rate (OCR) and extracellular acidification (ECAR), respectively. Additionally, we assessed the metabolite profile of 25 primary CLL cells (n=3 TRAF3 mut cells, n=22 TRAF3 wt cells) and their response to a library of drugs (CLL drugs and metabolic inhibitors), in the presence and absence of microenvironment (with and without HS-5 co-culture and IL-2 and CpG stimulation). Results: Metabolomic analysis of CRISPR/Cas9-edited CLL models showed an enrichment of metabolites involved in Warburg effect (pyruvate, phosphoenolpyruvate, and lactate), citric acid cycle (TCA- acetyl-CoA, α-ketoglutarate, succinate) and glutamate metabolism (α-ketoglutarate) in PGA1- TRAF3 mut cells. Moreover, PGA1- TRAF3 mut cells exhibited higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis was also slightly enhanced in the TRAF3 mut cells. Remarkably, primary CLL cells with TRAF3 mutations presented a higher concentration of phosphoenolpyruvate, glutamate and the TCA intermediates fumarate and malate, as well as higher levels of NADH and ATP, proving an increased energy production in these cells. Altogether, these results reveal that TRAF3 mut cells showed an enhanced mitochondrial glycolytic metabolism. Given the metabolic changes identified in TRAF3 mut cells, we subsequently assessed their OCR and ECAR after treatment with metabolic inhibitors. Unlike in WT cells, UK5099 administration (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) induced an increase in the maximal respiration of TRAF3 mut cells, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. Therefore, we tested the effect of C968 (glutaminolysis inhibitor), finding the same effect on TRAF3 mut cells, which reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis). Notably, the transcriptomic analysis revealed an upregulation of the glutamine transporter SLC1A4 (p-adj=0.007), what could be favoring glutamine uptake for an enhanced glutaminolysis. Interestingly, by blocking pyruvate import and glutaminolysis simultaneously, a decrease in TRAF3 mut CLL cells proliferation was observed (p=0.03), with equal levels of the previously dysregulated metabolites to those of the WT, which may indicate that this treatment sensitizes TRAF3 mut cells through its relieving effect on metabolic reprogramming. Moreover, primary CLL cells with TRAF3 mutations were also sensitive to the combination, in the presence and absence of microenvironment (p=0.01, p=0.04 respectively), demonstrating the potential of the combination of metabolic inhibitors as a new therapeutic approach in TRAF3 mut patients. Conclusion: Our work identifies a novel role of TRAF3 alterations in the CLL cellular metabolism. Specifically, TRAF3 inactivation induced an increased mitochondrial glycolytic activity and distinct metabolic dependencies in CLL cells, contributing to an enhanced metabolic plasticity (mitochondrial glycolysis/glutaminolysis) potentially targetable in CLL. Funding: PI21/00983 FEHH-GELLC(CPC), JYCL(ARS)

The Value of Neutrophil Gelatinase-Associated Lipocalin Receptor as a Novel Partner of CD38 in Chronic Lymphocytic Leukemia: From an Adverse Prognostic Factor to a Potential Pharmacological Target?

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of neoplastic B lymphocytes that escape death, and correlates with the expression of negative prognostic markers such as the CD38 antigen. Although certain new drugs approved by the US Food and Drug Administration improve the clinical outcome of CLL patients, drug resistance and disease relapse still occur. Like CD38, neutrophil gelatinase-associated lipocalin receptor (NGAL-R) is frequently overexpressed in CLL cells. Here, we evaluated the concomitant surface expression of NGAL-R and CD38 in leukemic blood cells from 52 CLL patients (37 untreated, 8 in clinical remission, and 7 relapsed). We provide evidence of a positive correlation between NGAL-R and CD38 levels both in the interpatient cohorts (p < 0.0001) and in individual patients, indicating a constitutive association of NGAL-R and CD38 at the cell level. Patients with progressing CLL showed a time-dependent increase in NGAL-R/CD38 levels. In treated CLL patients who achieved clinical remission, NGAL-R/CD38 levels were decreased, and were significantly lower than in the untreated and relapsed groups (p < 0.02). As NGAL-R and CD38 participate in CLL cell survival, envisioning their simultaneous inhibition with bispecific NGAL-R/CD38 antibodies capable of inducing leukemic cell death might provide therapeutic benefit for CLL patients.

The Role of Cholesterol in Chronic Lymphocytic Leukemia Development and Pathogenesis.

Cholesterol has many critical functions in cells. It is a key component of membranes and cell-signalling processes, and it functions as a chemical precursor in several biochemical pathways, such as Vitamin D and steroid synthesis. Cholesterol has also been implicated in the development and progression of various cancers, in which it is thought to promote cell proliferation, migration, and invasion. Chronic lymphocytic leukemia (CLL) is an example of a lipid-avid cancer that relies on lipid metabolism, rather than glycolysis, to fuel cell proliferation. However, data regarding the role of cholesterol in CLL are conflicting. Studies have shown that dyslipidaemia is more common among CLL patients than age-matched healthy controls, and that CLL patients who take cholesterol-lowering drugs, such as statins, appear to have improved survival rates. Therefore, defining the roles of cholesterol in CLL may highlight the importance of monitoring and managing hyperlipidaemia as part of the routine management of patients with CLL. In this review, we discuss the roles of cholesterol in the context of CLL by examining the literature concerning the trafficking, uptake, endogenous synthesis, and intracellular handling of this lipid. Data from clinical trials investigating various classes of cholesterol and lipid-lowering drugs in CLL are also discussed.

Trends in Medicare Spending on Oral Drugs for Chronic Lymphocytic Leukemia From 2014 to 2020

This cross-sectional study analyzes trends in Medicare Part D use and spending on oral-targeted drugs for chronic lymphocytic leukemia from 2014 to 2020.

Interferon gamma regulates a complex pro-survival signal network in chronic lymphocytic leukemia.

It is known that the microenvironmental cytokine interferon gamma (IFN-γ) provides a survival advantage for chronic lymphocytic leukemia (CLL) cells. However, the mechanisms involved in this effect have not been properly investigated. Herein, we conducted a comprehensive screening of the effects of IFN-γ on signaling pathways and gene expression profiles in CLL cells by using western blotting, real-time quantitative reverse transcription (RT-qPCR) and high-throughput RNA sequencing (RNA-seq). We found that IFN-γ not only activated the pro-survival signal transducer and activator of transcription 3 (STAT3), but also activated the protein kinase B and extracellular signal-regulated kinase signaling pathways. RNA-seq analysis showed that IFN-γ stimulation changed the expression profiles of more than 500 genes, with 391 being up-regulated and 123 down-regulated. These genes are involved in numerous biological processes, including anti-apoptosis, cell migration, and proliferation. IFN-γ significantly up-regulated the expression of CD38, BCL6, CXCL9, BCL2A1, SCOS3, IL-10, HGF, EGFR, THBS-1, FN1, and MUC1, which encode proteins potentially associated with disease progression, worse prognosis or poor response to treatment. Blocking janus kinases1/2 (JAK1/2) or STAT3 signal by specific inhibitors affected the expression of most genes, suggesting a pivotal role of the JAK1/2-STAT3 pathway in IFN-γ pro-survival effects in CLL. Our data demonstrate that IFN-γ regulates a complex pro-survival signal network in CLL through JAK1/2-STAT3, which provides a rational explanation for IFN-γ promoting CLL cells survival and drug resistance.

Effect of centralized procurement of drugs for chronic lymphocytic leukemia

Effect of centralized procurement of drugs for chronic lymphocytic leukemia

The Financial Toxicity of FDA-Drugs Approved for Malignant Hematology

Background: Many Food and Drug Administration (FDA) drugs (FDADs) are available now for patients with malignant hematology (MH). Still,the high prices of FDADs for MH affect the care of these patients and our health care system. Here, we estimated one monthly supply price (OMSP) for FDADs for MH approved between 2011 and 2021. Also, we reported the total cost (TC) of FDADs by multiplying OMSP times the assumed duration of therapy based on the available overall survival (OS) (OMSP x OS (months)). Methods: We performed a retrospective review of clinical trials (CTs) published from January 2011 to May 2021 that led to FDA approval of hematological malignancy drugs (Leukemias, lymphomas, Multiple Myeloma (MM), Myelodysplastic Syndrome (MDS), and Myeloproliferative Neoplasm (MPN)). The drugs investigated were selected using FDA Databases, 'Oncology (Cancer) / Hematologic Malignancies Approval Notifications' and 'Novel Drug Approvals'. CTs were also reviewed for demographic data on ClinicalTrials.gov (CT.gov) and Primary Literature on drug approval. FDADs were divided into disease groups: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), MDS+MPN, MM, and lymphoma. We compared prices between drug.com and UpToDate using the lowest price for each drug (the percentage change was low 0.18). We calculated the median of OMSP for each group, compared the median of OMSP between oral (PO), subcutaneous (SQ), and intravenous (IV) FDADs, and calculated TC for each group. For non-reached OS, we used 60 months to calculate TC. Results: Sixty-one FDADs were identified; 9,4,4,7 and 4 for AML, CML, ALL, CLL, and MDS+MPN, respectively. For MM and lymphoma, we identified 11 and 22, FDADs, respectively. The median of PO, SQ, and IV FDADs were 29, 4, and 30, respectively (some drugs were approved for more than one group). The median of OMSP for AML, CML, ALL, CLL, and MDS+MPN were $30,923 [range, $5,989-36,099], $19,343 [range, $20,834-22,570], $75,839 [range, $48,917-93,660] (excluding chimeric antigen receptor-T cell (CAR-T) therapy), $15,263 [range, $3,532- 23,091], and $15,284 [range, $8,060- 24,157], respectively. The median of OMSP for MM and lymphoma excluding CAR-T were $20,514 [range, $2,090-35,276 ] and $18,434 [range, $6,834-34,351], respectively. The median of OMSP for PO, SQ, and IV FDADs were $18,291 [range, $2,628-31,686], $18,370 [range, $6,934-22,583] and $24,691 [range, $2,090-93,660] (excluding CAR-T cell therapy), respectively. All FDADs for ALL patients were IV. The median OMSP for FDADs approved from (2011 to 2016) and (2016 to 2021) were $22,233 (range, $8,300-23,091) and $24,517 (range, $2,352-86,253), respectively. The median TC for AML, CML, ALL, CLL, and MDS+MPN were $351,327 (range, $196,380-791,041), $1,160,597 (range, $1,097,482-1,354,993), $547,091 (range, $244,587-$985,915), $971,160 (range, $564,720-3,456,000), and $1,114,038 (range, $255,502-1,449,447), respectively. OS was not reached for all CML drugs, in 5/7 (71%) of CLL drugs and 12/61 (34%) of all FDADs. The median TC for MM and lymphoma were $607,031 [range, $18,813-1,743,706 ] and $884,900 [range, $183,817-9,116,360], respectively. Excluding CAR-T drugs, The highest OMSP was for ALL but not TC due to the short median OS (10 months) (Figure 1). There were 5 CAR-T products approved between August 2017 and March 2021 (tisagenlecleucel, axicabtagene ciloleucel, lisocabtagene maraleucel, brexucabtagene autoleucel and idecabtagene vicleucel). The median cost was $432,055 (range, $373,000-475,000), and the estimated OMSP was $21,635 (range,$6,216-39,583). The median household monthly income was $6,558 in 2020 in the United States (US). In 2020, 8.6% of US population (28 million) did not have helath insurance at any point during the year. There are currently no generic drug products of these FDADs available in the US. Conclusion: The cost-effectiveness of CAR-T products is relatively similar to other FDADs. Patients with malignant hematologic disorders are affected by treatment-related financial harm. Although the costs used for this analyses may be modulated by many factors where the actual cost may not be what is reported in the sources used, the financial impact is still significant for many patients. There is a need for strategies to lower the costs. The value of drugs should be considered in formulating treatment guidelines. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Old and New Drugs for Chronic Lymphocytic Leukemia: Lights and Shadows of Real-World Evidence.

Several novel treatments for chronic lymphocytic leukemia (CLL) have been recently approved based on the results of randomized clinical trials. However, real-world evidence (RWE) is also requested before and after drug authorization in order to confirm safety and to provide data for health technology assessments. We conducted a scoping review of the available RWE for targeted treatments of CLL, namely ibrutinib, acalabrutinib, idelalisib, and venetoclax, as well as for chemoimmunotherapy (CIT). In particular, we searched studies published since 1 January 2010 and reported outcomes of the above treatments based on health databases, registries, or phase IV studies, including named-patient programs. We included both full papers and abstracts of studies presented at major meetings. Overall, 110 studies were selected and analyzed: 28,880 patients were treated with ibrutinib, 1424 with idelalisib, 751 with venetoclax, 496 with acalabrutinib, and 14,896 with CIT. Reported discontinuation rates were higher than in clinical trials, while effectiveness could not be indirectly compared with clinical trials since a detailed case mix, including cytogenetic risk factors, was partially available and propensity scores rarely applied. RWE on CLL can help to set realistic outcomes with novel treatments, however, real-world studies should be fostered, and available data shared.

Comparative Clinical Value of Pharmacologic Therapies for B-Cell Chronic Lymphocytic Leukemia: An Umbrella Analysis.

Several new drugs are progressively improving the life span of patients with B-cell chronic lymphocytic leukemia (CLL). However, the rapidly evolving standard of care precludes robust assessments of the incremental clinical value of further innovative drugs. Therefore, we systematically reviewed comparative evidence on newly authorized CLL drugs, as reported by standard and network meta-analyses (MA) published since 2016. Overall, 17 MAs addressed the relative survival or safety of naïve and/or refractory/relapsed (R/R) CLL patients. In R/R patients, therapies including BTK- and BCL2-inhibitors reported progression free survival (PFS) hazard ratios ranging from 0.08 to 0.24 (versus chemotherapy) and a significant advantage in overall survival (OS). In naïve patients, the PFS hazard ratios associated with four recent chemo-free therapies (obinutuzumab- and/or acalabrutinib-based) ranged from 0.11 to 0.61 versus current standard treatments (STs), without a significant OS advantage. Ten MAs addressed the risk of cardiovascular, bleeding, and infective events associated with BTK inhibitors, with some reporting a different relative safety in naïve and R/R patients. In conclusion, last-generation therapies for CLL consistently increase PFS, but not OS, and minimally decrease safety, as compared with STs. Based on available evidence, the patient-customized adoption of new therapies, rather than universal recommendations, seems desirable in CLL patients.

Druggable Molecular Pathways in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in adults, is characterized by a high degree of clinical heterogeneity that is influenced by the disease’s molecular complexity. The genes most frequently affected in CLL cluster into specific biological pathways, including B-cell receptor (BCR) signaling, apoptosis, NF-κB, and NOTCH1 signaling. BCR signaling and the apoptosis pathway have been exploited to design targeted medicines for CLL therapy. Consistently, molecules that selectively inhibit specific BCR components, namely Bruton tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) as well as inhibitors of BCL2, have revolutionized the therapeutic management of CLL patients. Several BTK inhibitors and PI3K inhibitors with different modes of action are currently used or are in development in advanced stage clinical trials. Moreover, the restoration of apoptosis by the BCL2 inhibitor venetoclax offers meaningful clinical activity with a fixed-duration scheme. Inhibitors of the BCR and of BCL2 are able to overcome the chemorefractoriness associated with high-risk genetic features, including TP53 disruption. Other signaling cascades involved in CLL pathogenesis, in particular NOTCH signaling and NF-kB signaling, already provide biomarkers for a precision medicine approach to CLL and may represent potential druggable targets for the future. The aim of the present review is to discuss the druggable pathways of CLL and to provide the biological background of the high efficacy of targeted biological drugs in CLL.

Targeting PAK1 Overcomes Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

Objective:Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance.Methods:An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining.Results:Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G).To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I).IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI<1) of this combinatorial therapy (Figure 1K).Conclusions:In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL) Patients Quality of Life (QoL): A Cross-Sectional Analysis of the Italian Experience in the Choice Study during the First Wave of the COVID-19 Pandemic

IntroductionAlthough plenty of data exists on efficacy and safety of CLL drugs, their impact on patients' Health-Related Quality of Life (HRQoL) is largely unknown (1-2). Documentation of drug safety via traditional use of adverse events (AE) in hematology is limited if not complemented with Patient-Reported Outcomes (PRO) measures (3-4). Incorporation of HRQoL PROs is now essential to better evaluate risk-benefit of new therapeutic approaches and it is also highly valued by regulatory stakeholders (5). Most PRO data currently available for CLL patients (pts) came from randomized controlled trial settings (6-7), hence limiting generalizability of findings to CLL real-life patients. CHOICE study was designed to investigate CLL patients' QoL and preference towards different treatment profiles through a Discrete Choice Experiment (DCE) methodology in Italy. Due to the timelines of the study, which started in February 2020, the related data offer an insight into patients' perception and worries during the first wave of the COVID-19 pandemic.MethodsThis cross sectional, multi-center, observational study included CLL patients, treatment naïve during the watch & wait period (W&W) or already TREATED (around 50% each, controlled at site level), who signed the informed consent for study participation. Exclusion criteria were inability to take oral drugs, cognitive disorders that could impair the comprehension of the questionnaires and concomitant treatment for other malignancies.Patients were asked to fill in the following HRQoL questionnaires: EQ-5D-5L, EORTC QLQ-C30 and QLQ CLL-16, as well as a DCE questionnaire, (described elsewhere). Each questionnaire was completed by the patient on a tablet - using an App specifically developed for the study.Results401 pts were enrolled in Italy in 16 hematology centers (Feb - July 2020); 199 W&W and 196 TREATED pts completed the questionnaires and were included in the evaluable population.Main patients' characteristics are shown in Table 1.73.7% of TREATED pts were ON-treatment (30.8% were in 1st-line, 69,2% in further lines) and 26.3% were OFF-treatment; the majority of pts (55,6%) were currently treated with a target therapy (Table1).The EQ-5D-5L questionnaire showed no significant differences between groups. In both groups more than 80% of pts reported low values (1 or 2, indicating no or small impact) on all items. Median VAS was 75 for the TREATED group and 80 for the W&W group (0-100; higher scores indicate higher QoL).QLQ C-30 / CLL-16 scores had very similar results between TREATED and W&W pts suggesting a limited impact of CLL on pts QoL. The median (IQR) QoL Scale was 83.3 (67- 83) for TREATED and 83.3 (67- 92) for W&W pts (0-100; all functional scales had high scores, that represent a better level of functioning; all symptoms' scales had low values, representing a less important symptomatology or problem, Figure 1). The main symptoms reported were fatigue, insomnia, pain, and dyspnea, while the main worry was for “future health” (Figure 1).Distribution of data was statistically different between the 2 groups only for the Role functioning Scale (p=0.024) and the Social Functioning Scale (p=0.003) of QLQ-C30 and for the Infection Scale (p<0.001) of QLQ CLL-16, always with slightly but significantly better results for the W&W group.ConclusionsCHOICE study helps to understand the CLL patients' mindset and feeling in the light of the COVID-19 pandemic impact on health care for this category of pts, highlighting their preferences and worries in a large cohort of pts in Italy, allowing a comparison between TREATED and W&W pts. The main limitation of the study was its cross-sectional design, which does not allow us to evaluate any change in QoL neither with respect to the impact of the pandemic, nor to the effects of the treatment, if any.CLL pts showed a good QoL, as confirmed by both EQ-5D-5L and EORTC QLQ C-30 / CLL-16 scores, with very similar results between TREATED and W&W pts (although slightly better results in the W&W vs TREATED group). The results of the present study are consistent with previous reports, and fatigue was the most reported symptom, while worry for future health was the most relevant score in CLL-16 questionnaire. Hospital accesses reduction that was detected during the pandemic might have influenced patients' response, as well as the extreme attention towards the danger of infections, and might have impacted patients' perception on future health. [Display omitted] DisclosuresTedeschi: Beigene: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; AbbVie: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau. Gozzetti: Janssen: Honoraria; AbbVie: Honoraria. Reda: Beigene: Consultancy; Astra Zeneca: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Gualberti: AbbVie: Current Employment. Malgieri: AbbVie: Current Employment. Finsinger: AbbVie: Current Employment.

New actionable targets and investigational drugs in chronic lymphocytic leukemia

SummaryThe treatment landscape of chronic lymphocytic leukemia (CLL) has shifted from chemotherapy-based approaches to targeted agents in the last decade. However, evolving drug resistance and accumulating toxicity remain challenges that still limit patients’ clinical outcomes. Furthermore, currently licensed targeted agents such as inhibitors of Bruton’s tyrosine kinase (BTK) and anti-apoptotic protein B‑cell lymphoma 2 (BCL2) do not adequately compensate for the poor clinical outcomes associated with high-risk genetics such as TP53 alterations. New insights into disease biology facilitated design and investigation of several new targeted agents with encouraging results in early clinical trials. This short review focuses on novel actionable targets and investigational drugs aimed at circumventing acquired resistance and avoiding accumulating toxicity.

The Gain of the Short Arm of Chromosome 2 (2pgain) Plays an Important Role in CLL Drug Resistance, and Has to be Systematically Considered before Deciding an Adapted Therapy

The Gain of the Short Arm of Chromosome 2 (2pgain) Plays an Important Role in CLL Drug Resistance, and Has to be Systematically Considered before Deciding an Adapted Therapy

A review of bioanalytical methods for chronic lymphocytic leukemia drugs and metabolites in biological matrices.

Quantitation of drugs used for the treatment of chronic lymphocytic leukemia in various biological matrices during both pre-clinical and clinical developments is very important, often in routine therapeutic drug monitoring. The first developed methods for quantitation were traditionally done on LC in combination with either UV or fluorescence detection. However, the emergence of LC with mass spectrometry in tandem in early 1990s has revolutionized the quantitation as it has provided better sensitivity and selectivity within a shorter run time; therefore it has become the choice of method for the analysis of various drugs. In this article, an overview of various bioanalytical methods (HPLC or LC-MS/MS) for the quantification of drugs for the treatment of chronic lymphocytic leukemia, along with applicability of these methods, is given.

Early Adoption and Outcomes of Ibrutinib As Treatment for Older Patients with Chronic Lymphocytic Leukemia (CLL): A Population-Based Study

Background: The availability of ibrutinib, an oral Bruton's tyrosine kinase inhibitor, has transformed the management of CLL. For older patients, first-line ibrutinib (± obinutuzumab) has shown advantage over chlorambucil (± obinutuzumab; Burger, NEJM 2015; Moreno, Lancet Oncol 2018) or bendamustine/rituximab (Woyach, NEJM 2019). However, its high cost raises concerns about potential disparities in access. Other concerns in older patients include the risk of atrial fibrillation (AFib), ventricular arrhythmias (VArr), or central nervous system hemorrhage (CNSh) associated with ibrutinib. We examined treatment patterns and outcomes among Medicare beneficiaries with CLL after the approval of ibrutinib in 2014. Methods: Using the linked SEER-Medicare registry, which covers ~34% of the US population, we identified Medicare beneficiaries with CLL diagnosed in 2007-2015, who had complete claims (including outpatient prescriptions), and who received systemic treatment in 2007-2016. We identified ibrutinib use as first or subsequent lines of therapy. We classified common first-line regimens as: 1) rituximab alone, 2) bendamustine ± CD20 antibody, 3) obinutuzumab ± chlorambucil, 4) other cytotoxic agents, and 5) ibrutinib (± CD20 antibody). We calculated total spending (observed in SEER-Medicare data) on CLL drugs per year, described duration of ibrutinib therapy, survival after starting ibrutinib, and the cumulative incidence function (CIF) for AFib, VArr, and CNSh during ibrutinib therapy. Using time-split survival analysis, we calculated the incidence rate ratio (IRR) for these events among patients starting ibrutinib compared with those managed without ibrutinib. All estimates report 95% confidence intervals (CI). Results: Ibrutinib was administered to 594 (18.2%) of 3,271 beneficiaries with CLL (at median age of 77 years [y]), including 196 in first line. The proportion of patients receiving ibrutinib as first-line therapy increased from 7% in 2014 to 36% in 2016 (Fig. A). As a result, Medicare spending on ibrutinib skyrocketed, surpassing all other CLL chemotherapeutics in 2016 (total $29.2M for ibrutinib, $18.5M for other drugs combined; Fig. B). Median gross cost of the first ibrutinib prescription was $9,301 (interquartile range [IQR], $8,635-10,233). Median patient's out-of-pocket (OOP) responsibility was $2,296 (IQR, $30-2,739), markedly lower for beneficiaries with the low-income subsidy ($4) than those without it ($2,538). Despite this variation, first-line ibrutinib use was not associated with any socio-demographic factor, including the low-income subsidy (P=.67 in χ2 test), age (P=.95), sex (P=.52), race/ethnicity (P=.31), or prevalent poverty (P=.35). Median duration of ibrutinib therapy was 13.6 months [mo.] (95%CI, 11.7-16.1), similar in first and subsequent line (P=.93, Fig. C). Median overall survival after starting ibrutinib was 30.3 mo. (95%CI, 22.3-not reached) in first line, and 24.6 mo. (95%CI, 21.6-28.7) in subsequent lines (Fig. D; P=.08). Median survival after stopping ibrutinib was 12.4 mo. (95%CI, 7.5-20.6) in first line, and 7.5 mo. (95%CI, 4.6-11.1) in subsequent lines (P=.06). The CIF of AFib on ibrutinib was 15.7% at 1 y (95%CI, 12.3-19.4), and 23.7% at 2 y (95%CI, 18.9-28.8; Fig. E). The incidence of AFib was significantly higher among patients receiving ibrutinib compared with other treatments (IRR, 1.65; 95%CI, 1.37-2.00). The CIF of VArr at 1 y was 4.4% (95%CI, 2.8-6.5%), also significantly increased (IRR, 1.62; 95%CI, 1.10-2.39). The CIF of CNSh at 1 y was 1.7% (95%CI, 0.8-3.0) (Fig. F), and significantly increased on ibrutinib (IRR, 4.48; 95%CI, 2.31-8.68). Among patients starting ibrutinib, 11.4% were on anticoagulants, 19.2% had prior atrial fibrillation, and 10.8% were on CYP3A4 inhibitors interacting with ibrutinib. Conclusions: Ibrutinib has been rapidly adopted for treatment of Medicare beneficiaries with CLL, resulting in major shifts in spending on CLL drugs. Despite high OOP costs, the use of first-line ibrutinib is not associated with sociodemographic characteristics, suggesting that therapeutic decisions are determined by clinical factors. The real-world survival of older patients on ibrutinib is poorer than seen in clinical trials, and rates of AFib, VArr and CNSh are higher. The use of ibrutinib in patients with pre-existing Afib or at risk for bleeding because of anticoagulation warrants particular attention. Disclosures Olszewski: Spectrum Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding; TG Therapeutics: Research Funding; Genentech: Research Funding. Davids:Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Research to Practice: Honoraria; AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees.

HIF-1α is over-expressed in leukemic cells from TP53-disrupted patients and is a promising therapeutic target in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the hypoxia-inducible factor 1 (HIF-1) regulates the response of tumor cells to hypoxia and their protective interactions with the leukemic microenvironment. In this study, we demonstrate that CLL cells from TP53-disrupted (TP53dis) patients have constitutively higher expression levels of the α-subunit of HIF-1 (HIF-1α) and increased HIF-1 transcriptional activity compared to the wild-type counterpart. In the TP53dis subset, HIF-1α upregulation is due to reduced expression of the HIF-1α ubiquitin ligase von Hippel-Lindau protein (pVHL). Hypoxia and stromal cells further enhance HIF-1α accumulation, independently of TP53 status. Hypoxia acts through the downmodulation of pVHL and the activation of the PI3K/AKT and RAS/ERK1-2 pathways, whereas stromal cells induce an increased activity of the RAS/ERK1-2, RHOA/RHOA kinase and PI3K/AKT pathways, without affecting pVHL expression. Interestingly, we observed that higher levels of HIF-1A mRNA correlate with a lower susceptibility of leukemic cells to spontaneous apoptosis, and associate with the fludarabine resistance that mainly characterizes TP53dis tumor cells. The HIF-1α inhibitor BAY87-2243 exerts cytotoxic effects toward leukemic cells, regardless of the TP53 status, and has anti-tumor activity in Em-TCL1 mice. BAY87-2243 also overcomes the constitutive fludarabine resistance of TP53dis leukemic cells and elicits a strongly synergistic cytotoxic effect in combination with ibrutinib, thus providing preclinical evidence to stimulate further investigation into use as a potential new drug in CLL.

Abstract 2072: Selinexor in combination with chemotherapy or idelalisib elicits a synergistic cytotoxic effect in primary CLL cells

Abstract Despite the therapeutic efficacy of new targeted drugs in chronic lymphocytic leukemia (CLL), treatment of high-risk patients remains an unmet clinical need. Tumor suppressor proteins (TSPs) and growth regulatory proteins (e.g. p53, p21, FoxO3a - the effector of Akt signal transduction, and IκB - the endogenous inhibitor of NF-κB) bind the nuclear export protein XPO1 and are carried through the nuclear pore complex into the cell cytoplasm. XPO1 overexpression plays a pathogenic role in CLL by mislocalizing TSPs and other cargoes in the cytoplasm. Selinexor is an oral inhibitor of XPO1, which is active as single agent in different hematologic malignancies. The purpose of this study was to evaluate the in vitro cytotoxic effects of selinexor, used in combination with chemotherapy drugs or with the PI3K inhibitor, idelalisib, against primary CLL cells. Specifically, this study aimed at identifying combination regimens that might overcome single agent resistance. Purified CLL cells from 29 patients were exposed to selinexor (Sel) in combination with fludarabine (F-ara-A), bendamustine (Ben) or idelalisib (Ide). Drug concentrations and exposure times were selected based on data obtained in an initial cohort of 5 patients. In selected experiments, the M2-10B4 murine stromal cell (SC) line was used in co-culture with CLL primary cells. Cell viability was analysed by Annexin-V/propidium Iodide immunostaining and flow cytometry. Combination index (CI) was determined using Calcusyn software. NF-kB and Akt activity was tested through an ELISA assay. After 72-hour culture, a significant decrease in CLL cell viability was observed when samples were treated with drug combinations (i.e. Sel 0.1 µM + F-ara-A 1 µM/F-ara-A 10 µM/Ben 30 µM/Ben 50 µM/Ide 0.1 µM/Ide 1 µM/ Ide 10 µM; Sel 1 µM + F-ara-A 1 µM/Ben 30 µM/Ben 50 µM) compared to the single compounds. The majority of combinations exerted synergistic cytotoxic effects. Interestingly, the addition of selinexor was effective in restoring the fludarabine sensitivity of CLL cells showing in vitro resistance to fludarabine. Sel was also capable to overcome the SC-mediated drug resistance, as shown by the significantly increased and synergistic cytotoxicity exerted by drug combinations, compared to single agents, toward leukemic cells co-cultured with SC. From the molecular standpoint, preliminary data showed that Sel significantly potentiated the inhibitory effect exerted by single-agent idelalisib on NF-kB and Akt activity. Our data demonstrate that the combination of Sel with chemotherapy or idelalisib has synergistic cytotoxic effects, also counteracting intrinsic or SC-mediated drug resistance. In vivo experiments in the Eμ-TCL-1 mouse model are currently ongoing to further corroborate the efficacy of selected drug combinations. Citation Format: Maria Todaro, Valentina Griggio, Chiara Salvetti, Chiara Riganti, Yosef Landesman, Mario Boccadoro, Candida Vitale, Marta Coscia. Selinexor in combination with chemotherapy or idelalisib elicits a synergistic cytotoxic effect in primary CLL cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2072.