Resistance to Bruton tyrosine kinase inhibition in chronic lymphocytic leukaemia and non-Hodgkin lymphoma.

Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma. However, primary and acquired resistance to BTKi can be seen due to a variety of mechanisms including tumour intrinsic and extrinsic mechanisms such as gene mutations, activation of bypass signalling pathways and tumour microenvironment. Herein, we provide an updated review of the key clinical data of BTKi treatment in CLL, mantle cell lymphoma, and diffuse large B-cell lymphoma (DLBCL). We incorporate the most recent findings regarding mechanisms of resistance to covalent and non-covalent inhibitors, including ibrutinib, acalabrutinib, zanubrutinib and pirtobrutinib. We also cover the clinical sensitivity of certain molecular subtypes of DLBCL to an ibrutinib-containing regimen. Lastly, we summarise ongoing clinical investigations aimed at overcoming resistance via use of BTKi-containing combined therapies or the novel non-covalent BTKi. The review article targets an audience of clinical practitioners, clinical investigators and translational researchers.

Multiple Genomic Alterations, Including a Novel AFF4::IRF1 Fusion Gene, in a Treatment-Refractory Blastic Plasmacytoid Dendritic-Cell Neoplasm: A Case Report and Literature Review.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with an aggressive clinical course and poor prognosis. The genetic abnormalities in BPDCN are heterogeneous; therefore, its molecular pathogenesis and the prognostic importance of genomic alterations associated with the disease are not well defined. Here we report a case of BPDCN with a novel AFF4::IRF1 fusion predicted to lead to a loss-of-function of the IRF1 tumor suppressor, somatic mutations of ASXL1, TET2, and MYD88, as well as multiple intrachromosomal deletions. The patient showed resistance to Tagraxofusp and Venetoclax, and he died about 16 months after diagnosis. Considering the predicted effect of the AFF4::IRF1 fusion on IRF1's antitumor effects and immune regulation, and the possibility of its relevance to the aggressive course observed in this case, we propose further evaluation of the clinical significance of this fusion in BPDCN in future cooperative group studies and the consideration of therapeutic strategies aimed at restoring IRF1-dependent antineoplastic effects in such cases.

Validation of a Molecular Diagnostic Test for Circulating Tumor DNA by Next-Gen Sequencing.

A modified version of the PGDx elioTM Plasma Resolve assay was validated as a laboratory-developed test (LDT) for clinical use in the Molecular Diagnostics Laboratory at Fox Chase Cancer Center. The test detects single nucleotide variants (SNVs) and small insertions and deletions (indels) in 33 target genes using fragmented genomic DNA extracted from plasma. The analytical performance of this assay was assessed with reference standard DNA and 29 samples from cancer patients and detected 66 SNVs and 23 indels. Using 50 ng of input DNA, the sensitivity was 95.5% to detect SNVs at 0.5% allele frequency, and the specificity was 92.3%. The sensitivity to detect indels at 1% allele frequency was 70.4%. A cutoff of 0.25% variant allele frequency (VAF) was set up for diagnostic reporting. An inter-laboratory study of concordance with an orthologous test resulted in a positive percent agreement (PPA) of 91.7%.

Targeting the Hsp90-associated viral oncoproteome in gammaherpesvirus-associated malignancies.

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.

Characterization of ibrutinib-sensitive and -resistant mantle lymphoma cells.

Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one-third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib-sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti-tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach.

Acute myeloid leukemia with a novel AKAP9::PDGFRA fusion transformed from essential thrombocythemia: A case report and mini review.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy associated with various combinations of gene mutations, epigenetic abnormalities, and chromosome rearrangement-related gene fusions. Despite the significant degree of heterogeneity in its pathogenesis, many gene fusions and point mutations are recurrent in AML and have been employed in risk stratification over the last several decades. Gene fusions have long been recognized for understanding tumorigenesis and their proven roles in clinical diagnosis and targeted therapies. Advances in DNA sequencing technologies and computational biology have contributed significantly to the detection of known fusion genes as well as for the discovery of novel ones. Several recurring gene fusions in AML have been linked to prognosis, treatment response, and disease progression. In this report, we present a case with a long history of essential thrombocythemia and hallmark CALR mutation transforming to AML characterized by a previously unreported AKAP9::PDGFRA fusion gene. We propose mechanisms by which this fusion may contribute to the pathogenesis of AML and its potential as a molecular target for tyrosine kinase inhibitors.

SYK inhibition and response prediction in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G(1)-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

Isochromosome 7p, i(7)(p10): A rare AML, myelodysplasia-related entity.

We describe genomic findings in an AML case with isochromosome 7p, i(7)(p10), in which SNP array analysis uncovered an additional 7.07-Mb 20q deletion not detected by karyotyping. Several AML cases with i(7)(p10) as an isolated cytogenetic finding have been previously reported. Based on consequent loss of 7q, we propose that AML with i(7)(p10) represents a distinct entity belonging in the WHO group -7/7q-, which represents one of the genetic abnormalities defining AML, myelodysplasia-related. Additionally, the focal del(20q) identified here adds support for a specific common region of deletion in 20q in myeloid malignancies, implicating a small number of candidate genes.

Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

Splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms: heterogeneous morphology and cytological composition.

We studied 24 spleens with extramedullary hematopoietic proliferation (EMHP), a key feature of advanced-stage Philadelphia chromosome-negative myeloproliferative neoplasms, obtained from 24 patients (14 primary myelofibrosis, 7 polycythemia vera and 3 unclassifiable). Hematoxylin and eosin, reticulin and trichrome stains, and immunohistochemical stains for myeloperoxidase, glycophorin-C, CD42b, CD34, CD117, CD8, nerve growth factor receptor and smooth muscle actin were evaluated. Clinical information was correlated with the morphological findings. Three distinct histological patterns of EMHP were recognized: diffuse (12), nodular (5), and mixed-nodular and diffuse (7). The preponderant lineage was granulocytic in diffuse, trilineage in nodular and erythroid in mixed EMHP. Erythropoiesis was largely intravascular, granulopoiesis was within the splenic cords and megakaryopoiesis was observed in both locations. The stromal changes paralleled the histological pattern with preservation of the splenic stromal and vascular architecture in the diffuse areas as opposed to areas of nodular EMHP. The morphological features of the splenic EMHP did not correlate with specific subtypes of myeloproliferative neoplasms. The mean duration of follow-up from initial diagnosis was 80 months. A total of 15 of the 24 patients died of disease: 8 of 12 (67%) with diffuse, 2 of 5 (40%) with nodular and 5 of 7 (71%) with mixed growth patterns. The mean duration from diagnosis to splenectomy was shorter in patients with diffuse (83 months) as compared with those with nodular EMHP (127 months). Our study demonstrates that splenic extramedullary hematopoietic proliferation in Philadelphia chromosome-negative myeloproliferative neoplasms shows distinct histological patterns that do not correlate with disease subtypes, but appear to suggest a trend between the histological patterns and clinical behavior. These results suggest a different biology of the disease in the nodular and diffuse extramedullary hematopoietic proliferation groups.

Decrease in JAK2 V617F allele burden is not a prerequisite to clinical response in patients with polycythemia vera.

BACKGROUND: Although reduction in the JAK2(V617F) allele burden (%V617F) has been suggested as a criterion for defining disease response to cytoreductive therapy in polycythemia vera, its value as a response monitor is unclear. The purpose of this study is to determine whether a reduction in %V617F in polycythemia vera is a prerequisite to achieving hematologic remission in response to cytoreductive therapy. DESIGN AND METHODS: We compared the clinical and hematologic responses to change in %V617F (molecular response) in 73 patients with polycythemia vera treated with either interferon (rIFNα-2b: 28, Peg-rIFNα-2a: 18) or non-interferon drugs (n=27), which included hydroxyurea (n=8), imatinib (n=12), dasatinib (n=5), busulfan (n=1), and radioactive phosphorus (n=1). Hematologic response evaluation employed Polycythemia Vera Study Group criteria, and molecular response evaluation, European Leukemia Net criteria. RESULTS: Of the 46 treated with interferon, 41 (89.1%) had a hematologic response, whereas only 7 (15.2%) had a partial molecular response. Of the 27 who received non-interferon treatments, 16 (59.3%) had a hematologic response, but only 2 (7.4%) had a molecular response. Median duration of follow up was 2.8 years. Statistical agreement between hematologic response and molecular response was poor in all treatment groups. CONCLUSIONS: Generally, hematologic response was not accompanied by molecular response. Therefore, a quantitative change in %V617F is not required for clinical response in patients with polycythemia vera.

Epigenetic down-regulation of the tumor suppressor gene PRDM1/Blimp-1 in diffuse large B cell lymphomas: a potential role of the microRNA let-7.

PRDM1/Blimp-1, a master regulator for B cell terminal differentiation, is a putative tumor suppressor in diffuse large B cell lymphomas (DLBCL). Inactivating mutations of PRDM1 have been previously identified in a subset of nongerminal center B cell-like (GCB) DLBCL. We investigated the presence of alternative mechanisms of down-regulating PRDM1 in a cohort of 25 primary DLBCL and six DLBCL cell lines. While some DLBCL, predominantly the GCB-type, showed low levels of both PRDM1alpha mRNA and protein, presumably as a result of direct transcription repression, discordant expressions between the two were identified in a subset of DLBCL without PRDM1 mutations, the primarily non-GCB type, consistent with translational down-regulation. This subset of DLBCL exhibits relatively high PRDM1alpha mRNA levels but low levels of PRDM1. Data obtained from expression analysis, luciferase reporter assays, and transfection experiments support a role of targeting of PRDM1 by microRNA let-7 family in mediating this down-regulation. Let-7, in particular let-7b, is overexpressed in DLBCL relative to normal GCB cells, suggesting that it is deregulated. Thus, abnormal epigenetic down-regulation of PRDM1 by let-7 and other microRNAs may represent an alternative mechanism of reducing normal PRDM1 function in a subset of DLBCL with relatively high PRDM1alpha mRNA expression and unmutated PRDM1. These findings provide further evidence for an important role of impairment of terminal B cell differentiation in DLBCL pathogenesis.

Ibrutinib and venetoclax target distinct subpopulations of CLL cells: implication for residual disease eradication.

Ibrutinib inhibits Bruton tyrosine kinase while venetoclax is a specific inhibitor of the anti-apoptotic protein BCL2. Both drugs are highly effective as monotherapy against chronic lymphocytic leukemia (CLL), and clinical trials using the combination therapy have produced remarkable results in terms of rate of complete remission and frequency of undetectable minimal residual disease. However, the laboratory rationale behind the success of the drug combination is still lacking. A better understanding of how these two drugs synergize would eventually help develop other rational combination strategies. Using an ex vivo model that promotes CLL proliferation, we show that modeled ibrutinib proliferative responses, but not viability responses, correlate well with patients' actual clinical responses. Importantly, we demonstrate for the first time that ibrutinib and venetoclax act on distinct CLL subpopulations that have different proliferative capacities. While the dividing subpopulation of CLL responds to ibrutinib, the resting subpopulation preferentially responds to venetoclax. The combination of these targeted therapies effectively reduced both the resting and dividing subpopulations in most cases. Our laboratory findings help explain several clinical observations and contribute to the understanding of tumor dynamics. Additionally, our proliferation model may be used to identify novel drug combinations with the potential of eradicating residual disease.

Inhibition of B-cell receptor signaling disrupts cell adhesion in mantle cell lymphoma via RAC2.

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

Colorectal cancer expression of peroxisome proliferator-activated receptor gamma (PPARG, PPARgamma) is associated with good prognosis.

BACKGROUND & AIMS: The peroxisome proliferator-activated receptor gamma (PPARG, PPARgamma) is a nuclear receptor that regulates expression of mediators of lipid metabolism and the inflammatory response. There is controversy over the pro-oncogenic or antioncogenic effects of PPARG, and little is known about its prognostic significance in colon cancer. METHODS: Among 470 patients with colorectal cancer (stages I-IV) identified in 2 independent prospective cohorts, PPARG expression was detected in 102 tumors (22%) by immunohistochemistry. Cox proportional hazards models were used to compute hazard ratios (HRs) of colorectal cancer-specific and overall mortalities, adjusted for patient characteristics and molecular features including cyclooxygenase 2, fatty acid synthase, KRAS, BRAF, PIK3CA, p53, p21, beta-catenin, LINE-1 hypomethylation, microsatellite instability (MSI), and the CpG island methylation phenotype (CIMP). RESULTS: Compared with patients with PPARG-negative tumors, patients with PPARG-positive tumors had significantly lower overall mortality, determined by Kaplan-Meier analysis (P=.0047), univariate Cox regression (HR, 0.55; 95% confidence interval [CI], 0.37-0.84; P=.0053), and multivariate analysis (adjusted HR, 0.43; 95% CI, 0.27-0.69; P=.0004). Patients with PPARG-positive tumors experienced lower colorectal cancer-specific mortality (adjusted HR, 0.44; 95% CI, 0.25-0.79; P=.0054). The relationship between PPARG and lower mortality did not appear to be significantly modified by MSI, CIMP, LINE-1, or the other clinical and molecular variables examined (all P(interaction)>.05). CONCLUSIONS: Tumor expression of PPARG is independently associated with longer survival of patients. PPARG expression appears to mark an indolent subset of colorectal cancers.

Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials.

An international basis for comparison of BCR-ABL mRNA levels is required for the common interpretation of data derived from individual laboratories. This will aid clinical decisions for individual patients with chronic myeloid leukemia (CML) and assist interpretation of results from clinical studies. We aligned BCR-ABL values generated by 38 laboratories to an international scale (IS) where a major molecular response (MMR) is 0.1% or less. Alignment was achieved by application of laboratory-specific conversion factors calculated by comparisons performed with patient samples against a reference method. A validation procedure was completed for 19 methods. We determined performance characteristics (bias and precision) for consistent interpretation of MMR after IS conversion. When methods achieved an average BCR-ABL difference of plus or minus 1.2-fold from the reference method and 95% limits of agreement within plus or minus 5-fold, the MMR concordance was 91%. These criteria were met by 58% of methods. When not met, the MMR concordance was 74% or less. However, irrespective of precision, when the bias was plus or minus 1.2-fold as achieved by 89% of methods, there was good agreement between the overall MMR rates. This indicates that the IS can deliver accurate comparison of molecular response rates between clinical trials when measured by different laboratories.

Prognostic and Predictive Molecular Biomarkers in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a malignancy of B cells with a variable clinical course. Prognostication is important to place patients into different risk categories for guiding decisions on clinical management, to treat or not to treat. Although several clinical, cytogenetic, and molecular parameters have been established, in the past decade, a tremendous understanding of molecular lesions has been obtained with the advent of high-throughput sequencing. Meanwhile, rapid advances in the understanding of the CLL oncogenic pathways have led to the development of small-molecule targeting signal transducers, Bruton tyrosine kinase and phosphatidylinositol 3-kinase, as well as anti-apoptotic protein BCL2 apoptosis regulator. After an initial response to these targeted therapies, some patients develop resistance and experience disease progression. Novel gene mutations have been identified that account for some of the drug resistance mechanisms. This article focuses on the prognostic and predictive molecular biomarkers in CLL relevant to the molecular pathology practice, beginning with a review of well-established prognostic markers that have already been incorporated into major clinical guidelines, which will be followed by a discussion of emerging biomarkers that are expected to impact clinical practice soon in the future. Special emphasis will be put on predictive biomarkers related to newer targeted therapies in hopes that this review will serve as a useful reference for molecular diagnostic professionals, clinicians, as well as laboratory investigators and trainees.

Cerdulatinib Pharmacodynamics and Relationships to Tumor Response Following Oral Dosing in Patients with Relapsed/Refractory B-cell Malignancies.

PURPOSE: Preclinical studies suggest SYK and JAK contribute to tumor-intrinsic and microenvironment-derived survival signals. The pharmacodynamics of cerdulatinib, a dual SYK/JAK inhibitor, and associations with tumor response were investigated. PATIENTS AND METHODS: In a phase I dose-escalation study in adults with relapsed/refractory B-cell malignancies, cerdulatinib was administered orally to sequential dose-escalation cohorts using once-daily or twice-daily schedules. The study enrolled 8 patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), 13 with follicular lymphoma, 16 with diffuse large B-cell lymphoma (DLBCL), and 6 with mantle cell lymphoma. Correlation of tumor response with pharmacodynamic markers was determined in patients with meaningful clinical responses. RESULTS: Following cerdulatinib administration, complete SYK and JAK pathway inhibition was achieved in whole blood of patients at tolerated exposures. Target inhibition correlated with serum cerdulatinib concentration, and IC50 values against B-cell antigen receptor (BCR), IL2, IL4, and IL6 signaling pathways were 0.27 to 1.11 µmol/L, depending on the phosphorylation event. Significant correlations were observed between SYK and JAK pathway inhibition and tumor response. Serum inflammation markers were reduced by cerdulatinib, and several significantly correlated with tumor response. Diminished expression of CD69 and CD86 (B-cell activation markers), CD5 (negative regulator of BCR signaling), and enhanced expression of CXCR4 were observed in 2 patients with CLL, consistent with BCR and IL4 suppression and loss of proliferative capacity. CONCLUSIONS: Cerdulatinib potently and selectively inhibited SYK/JAK signaling at tolerated exposures in patients with relapsed/refractory B-cell malignancies. The extent of target inhibition in whole-blood assays and suppression of inflammation correlated with tumor response. (ClinicalTrials.gov ID:NCT01994382).

Metabolic Detection of Bruton's Tyrosine Kinase Inhibition in Mantle Cell Lymphoma Cells.

Current methods to evaluate effects of kinase inhibitors in cancer are suboptimal. Analysis of changes in cancer metabolism in response to the inhibitors creates an opportunity for better understanding of the interplay between cell signaling and metabolism and, from the translational perspective, potential early evaluation of response to the inhibitors as well as treatment optimization. We performed genomic, metabolomic, and fluxomic analyses to evaluate the mechanism of action of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) in mantle cell lymphoma (MCL) cells. Our comprehensive analysis of the data generated by these diverse technologies revealed that IBR profoundly affected key metabolic pathways in IBR-sensitive cells including glycolysis, pentose phosphate pathway, TCA cycle, and glutaminolysis while having much less effects on IBR-poorly responsive cells. Changes in 1H magnetic resonance spectroscopy (MRS)-detectable lactate and alanine concentrations emerged as promising biomarkers of response and resistance to IBR as demonstrated from experiments on various MCL cell lines. The metabolic network analysis on the 13C MRS and 13C LC/MS experimental data provided quantitative estimates of various intracellular fluxes and energy contributions. Glutaminolysis contributed over 50% of mitochondrial ATP production. Administration of the glutaminase inhibitor CB-839 induced growth suppression of the IBR-poorly responsive cells. IMPLICATIONS: Our study demonstrates application of the advanced metabolomic/fluxomic techniques for comprehensive, precise, and prompt evaluations of the effects of kinase inhibition in MCL cells and has strong translational implications by potentially permitting early evaluation of cancer patient response versus resistance to kinase inhibitors and on design of novel therapies for overcoming the resistance.