Associations of coffee/caffeine consumption with postmenopausal breast cancer risk and their interactions with postmenopausal hormone use.

PURPOSE: We investigated the association of coffee and caffeine with breast cancer (BCa) risk, overall and by ER/PR status. We also examined potential interactions of coffee and caffeine with postmenopausal hormone use. METHODS: Our study included 77,688 postmenopausal participants from the Women's Health Initiative observational study cohort without a history of any cancer at baseline (except non-melanoma skin) and with valid Food Frequency Questionnaire data and complete data on dietary caffeine. Regular coffee (none, 1, 2-3, 4-5, and ≥ 6 cups/day) and caffeine (tertiles) were assessed at baseline. Information on BCa risk factors was collected at baseline. The associations were examined using survival analysis, accounting for death as a competing risk. RESULTS: The median follow-up time for our cohort was 18.3 years. During the follow-up, 5005 women developed invasive breast cancer. In multivariable analysis, coffee was not associated with the overall invasive BCa risk. Higher caffeine intake was mildly associated with increased BCa risk (2nd vs. 1st tertile SHR = 1.10, 95% CI 1.03-1.18, 3rd vs. 1st tertile SHR-1.05, 95% CI 0.98-1.13, overall p = 0.03). We found no interaction of coffee/caffeine with postmenopausal hormone use (p interaction = 0.44 and 0.42, respectively). In the exploratory analysis by ER/PR status, we found a positive association of caffeine with ER+ /PR+ BCa (2nd vs. 1st tertile SHR = 1.17, 95% CI 1.07-1.28, 3rd vs. 1st tertile SHR = 1.13, 95% CI 1.03-1.24, overall p = 0.002); no associations were observed for ER-/PR- tumors. Coffee was not associated with the risk of ER+ /PR+ or ER-/PR- tumors. CONCLUSION: We found no associations of coffee with BCa risk, overall and for ER/PR-defined tumor subtypes. The higher caffeine consumption was mildly and positively associated with the overall BCa risk and with ER+ /PR+ tumors.

Noncovalent inhibition of C481S Bruton tyrosine kinase by GDC-0853: a new treatment strategy for ibrutinib-resistant CLL.

The clinical success of ibrutinib validates Bruton tyrosine kinase (BTK) inhibition as an effective strategy for treating hematologic malignancies, including chronic lymphocytic leukemia (CLL). Despite ibrutinib's ability to produce durable remissions in patients, acquired resistance can develop, mostly commonly by mutation of C481 of BTK in the ibrutinib binding site. Here, we characterize a novel BTK inhibitor, GDC-0853, to evaluate its preclinical efficacy in ibrutinib-naive and ibrutinib-resistant CLL. GDC-0853 is unique among reported BTK inhibitors in that it does not rely upon covalent reaction with C481 to stabilize its occupancy within BTK's adenosine triphosphate binding site. As with ibrutinib, GDC-0853 potently reduces B-cell receptor signaling, viability, NF-κB-dependent transcription, activation, and migration in treatment naïve CLL cells. We found that GDC-0853 also inhibits the most commonly reported ibrutinib-resistant BTK mutant (C481S) both in a biochemical enzyme activity assay and in a stably transfected 293T cell line and maintains cytotoxicity against patient CLL cells harboring C481S BTK mutations. Additionally, GDC-0853 does not inhibit endothelial growth factor receptor or ITK, 2 alternative targets of ibrutinib that are likely responsible for some adverse events and may reduce the efficacy of ibrutinib-antibody combinations, respectively. Our results using GDC-0853 indicate that noncovalent, selective BTK inhibition may be effective in CLL either as monotherapy or in combination with therapeutic antibodies, especially among the emerging population of patients with acquired resistance to ibrutinib therapy.

Medical dissolution therapy for the treatment of uric acid nephrolithiasis.

INTRODUCTION: Uric acid (UA) nephrolithiasis represents 10% of kidney stones in the US with low urine pH and high saturation of UA as the main risk factors for stone development. Dissolution therapy for UA kidney stones via urinary alkalization has been described as a treatment option. We present our experience in treating UA nephrolithiasis with medical dissolution therapy. METHODS: A retrospective review was performed of UA stone patients referred for surgery but treated with dissolution therapy between July 2007 and July 2016. Patients were identified using ICD-9 codes. Patients were treated with potassium citrate alone or in combination with allopurinol. Serial imaging and urine pH were obtained at follow-up. Demographics, aggregate stone size, time to stone clearance, urine pH (office dip), and complications were recorded. RESULTS OBTAINED: Twenty-four patients (14 men and 10 women) were identified that started medical dissolution therapy for UA nephrolithiasis after initial referral for surgical management. Three patients (13%) did not tolerate the initiation of dissolution therapy and discontinued this treatment. Of the 21 patients that were maintained on dissolution therapy, 14 patients (67%) showed complete resolution of nephrolithiasis and 7 patients (33%) showed partial reduction. Patients with partial response had a mean reduction in stone burden of 68%. There were 3 recorded complications (UTI, GI upset with therapy, and throat irritation) and 4 recorded stone recurrences among these 21 patients. CONCLUSION: Based on our study population, medical dissolution therapy is a well-tolerated, non-invasive option for UA nephrolithiasis.

Targeting the C481S Ibrutinib-Resistance Mutation in Bruton's Tyrosine Kinase Using PROTAC-Mediated Degradation.

Inhibition of Bruton's tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet >80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist, and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates on the basis of its potency to induce BTK knockdown. MT-802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib does and retains an equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the C481S mutation, MT-802 is able to reduce the pool of active, phosphorylated BTK whereas ibrutinib cannot. Collectively, these data provide a basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of C481S mutant CLL.

Targeting BTK through microRNA in chronic lymphocytic leukemia.

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Cardiovascular disease mortality among women with endometrial cancer in the Iowa Women's Health Study.

PURPOSE: Obesity is associated with endometrial cancer (EC) development and cardiovascular disease (CVD) mortality. As the number of obese EC survivors continues to increase, an examination of CVD mortality in this vulnerable population is warranted. METHODS: In the Iowa Women's Health Study (1986-2011), we examined CVD mortality among 552 women with EC compared with 2,352 age- and body mass index-matched women without EC (controls). Hazard ratios (HRs) and 95% confidence intervals (CIs) for CVD mortality were estimated using multivariable-adjusted Cox proportional hazards regression models stratified by an indicator for match set. RESULTS: Compared to controls, women with EC more often reported a history of diabetes, hypertension, and never smoking. Compared with controls, women with EC had lower CVD mortality (HR 0.75, 95% CI 0.56-0.99), and higher all-cause mortality (HR 1.50, 95% CI 1.30-1.74). CONCLUSIONS: Although some CVD risk factors were more common in women with versus without EC, CVD mortality was lower among the former group. Additional well-adjusted analyses with larger study populations are needed to understand interactions between CVD risk factors with CVD mortality among EC survivors. The CVD risk factor profile of EC survivors warrants emphasis on cardiovascular health.

Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling.

Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia.

Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells.

Aberrant regulation of endogenous survival pathways plays a major role in progression of chronic lymphocytic leukemia (CLL). Signaling via conjugation of surface receptors within the tumor environmental niche activates survival and proliferation pathways in CLL. Of these, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway appears to be pivotal to support CLL pathogenesis, and pharmacologic inhibitors targeting this axis have shown clinical activity. Here we investigate OSU-T315, a compound that disrupts the PI3K/AKT pathway in a novel manner. Dose-dependent selective cytotoxicity by OSU-T315 is noted in both CLL-derived cell lines and primary CLL cells relative to normal lymphocytes. In contrast to the highly successful Bruton's tyrosine kinase and PI3K inhibitors that inhibit B-cell receptor (BCR) signaling pathway at proximal kinases, OSU-T315 directly abrogates AKT activation by preventing translocation of AKT into lipid rafts without altering the activation of receptor-associated kinases. Through this mechanism, the agent triggers caspase-dependent apoptosis in CLL by suppressing BCR, CD49d, CD40, and Toll-like receptor 9-mediated AKT activation in an integrin-linked kinase-independent manner. In vivo, OSU-T315 attains pharmacologically active drug levels and significantly prolongs survival in the TCL1 mouse model. Together, our findings indicate a novel mechanism of action of OSU-T315 with potential therapeutic application in CLL.

Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation.

Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib's capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2(R665W) confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance.

IPI-145 antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) displays constitutive phosphatidylinositol 3-kinase (PI3K) activation resulting from aberrant regulation of B-cell receptor (BCR) signaling. Previous studies have shown that an oral PI3K p110δ inhibitor idelalisib exhibits promising activity in CLL. Here, we demonstrate that a dual PI3K p110δ and p110γ inhibitor, IPI-145, antagonizes BCR crosslinking activated prosurvival signals in primary CLL cells. IPI-145 causes direct killing in primary CLL cells in a dose- and time-dependent fashion, but does not generate direct cytotoxicity to normal B cells. However, IPI-145 does reduce the viability of normal T and natural killer cells and decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Furthermore, IPI-145 overcomes the ibrutinib resistance resulting from treatment-induced BTK C481S mutation. Collectively, these studies provide rationale for ongoing clinical evaluation of IPI-145 as a targeted therapy for CLL and related B-cell lymphoproliferative disorders.

PKC-ß as a therapeutic target in CLL: PKC inhibitor AEB071 demonstrates preclinical activity in CLL.

Targeting B-cell receptor (BCR) signaling in chronic lymphocytic leukemia (CLL) has been successful with durable remissions observed with several targeted therapeutics. Protein kinase C-ß (PKC-ß) is immediately downstream of BCR and has been shown to be essential to CLL cell survival and proliferation in vivo. We therefore evaluated sotrastaurin (AEB071), an orally administered potent PKC inhibitor, on CLL cell survival both in vitro and in vivo. AEB071 shows selective cytotoxicity against B-CLL cells in a dose-dependent manner. Additionally, AEB071 attenuates BCR-mediated survival pathways, inhibits CpG-induced survival and proliferation of CLL cells in vitro, and effectively blocks microenvironment-mediated survival signaling pathways in primary CLL cells. Furthermore, AEB071 alters ß-catenin expression, resulting in decreased downstream transcriptional genes as c-Myc, Cyclin D1, and CD44. Lastly, our preliminary in vivo studies indicate beneficial antitumor properties of AEB071 in CLL. Taken together, our results indicate that targeting PKC-ß has the potential to disrupt signaling from the microenvironment contributing to CLL cell survival and potentially drug resistance. Future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients are warranted.

Comparison of 2 Population Health Management Approaches to Increase Vitamin B12 Monitoring in Patients Taking Metformin.

BACKGROUND: Population health management uses proactive, targeted interventions to improve health outcomes. OBJECTIVES: To compare the effectiveness of and time required for 2 pharmacist-driven population health management interventions to improve vitamin B12 monitoring in patients taking metformin. METHODS: Physicians were randomized to 1 of 2 population health management interventions. For all patients of physicians assigned to the patient portal intervention, a pharmacist communicated need for vitamin B12 monitoring directly to the patient using an electronic patient portal. For all patients of physicians assigned to the office visit intervention, a pharmacist communicated monitoring recommendations to the physician prior to a scheduled office visit through the electronic health record. The proportion of patients in each group who received vitamin B12 monitoring 30 days after the intervention was quantified. RESULTS: A total of 489 patients of 26 physicians within 5 general internal medicine clinics who had taken metformin for at least 1 year and had not received vitamin B12 monitoring within the past year were identified. The intervention delivered as part of an office visit resulted in increased serum vitamin B12 monitoring compared with the intervention using electronic communication through a patient portal (odds ratio = 4.05; 95% CI = 1.22, 13.46; P = 0.03). The office visit intervention and the patient portal intervention took an average of 8.2 and 0.9 minutes per patient, respectively. CONCLUSIONS: Population health management completed during the course of office visits was more effective at increasing vitamin B12 monitoring and required more time than an intervention delivered through an electronic patient portal.

Tubal Ligation and Risk of Endometrial Cancer: Findings From the Women's Health Initiative.

OBJECTIVE: Bilateral tubal ligation (BTL) is a common form of birth control in the United States. There are limited, contradictory data examining BTL and the risk of endometrial cancer and none examining type I and type II cancers separately. We investigated the association between BTL and endometrial cancer risk using the Women's Health Initiative (WHI) Observational and Dietary Modification Studies. METHODS: Demographic information and history of BTL were obtained from the baseline questionnaires from 76,483 WHI participants in the Observational and Dietary Modification Studies. Univariable and multivariable models were used to examine the association of BTL with type I and type II endometrial cancers. RESULTS: A total of 1137 women were diagnosed with incident endometrial cancer (972 type I and 128 type II) during a mean follow-up of 11.3 years. Overall, 14,499 (19%) women had undergone BTL. There were no statistically significant associations noted between BTL and age at BTL for type I or type II cancers. CONCLUSIONS: We examined the largest patient cohort to date in an effort to determine the impact of BTL on endometrial cancer risk. In the WHI trial, we observed no overall effect of BTL on the risk of type I or type II endometrial cancer, suggesting that patients undergoing this popular birth control method likely do not have an associated change in their baseline risk for endometrial cancer.

Statins and breast cancer stage and mortality in the Women's Health Initiative.

PURPOSE: To evaluate the association between statins and breast cancer stage and mortality in the Women's Health Initiative. METHODS: The study population included 128,675 postmenopausal women aged 50-79 years, out of which there were 7,883 newly diagnosed cases of in situ (19%), local (61%)-, regional (19%)- and distant (1%)-stage breast cancer and 401 deaths due to breast cancer after an average of 11.5 (SD = 3.7) years of follow-up. Stage was coded using SEER criteria and was stratified into early (in situ and local)- versus late (regional and distant)-stage disease. Information on statins and other risk factors were collected by self- and interviewer-administered questionnaires. Cause of death was based on medical record review. Multivariable-adjusted hazards ratios (HR) and 95% confidence intervals (CIs) evaluating the relationship between statin use (at baseline only and in a time-dependent manner) and diagnosis of late-stage breast cancer and breast cancer-specific mortality were computed from Cox proportional hazards analyses after adjusting for appropriate confounders. RESULTS: Statins were used by 10,474 women (8%) at baseline. In the multivariable-adjusted time-dependent model, use of lipophilic statins was associated with a reduction in diagnosis of late-stage breast cancer (HR 0.80, 95% CI 0.64-0.98, p = 0.035) which was also significant among women with estrogen receptor-positive disease (HR 0.72, 95% CI 0.56-0.93, p = 0.012). Breast cancer mortality was marginally lower in statin users compared with nonusers (HR 0.59, 95 % CI 0.32-1.06, p = 0.075). CONCLUSIONS: Prior statin use is associated with lower breast cancer stage at diagnosis.

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes.

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

Impact of induction immunosuppression on survival in heart transplant recipients: a contemporary analysis of agents.

INTRODUCTION: The impact of induction immunosuppression on long-term survival in heart transplant recipients is unclear. Over the past three decades, practices have varied as induction agents have changed and experiences grew. We sought to evaluate the effect of contemporary induction immunosuppression agents in heart transplant recipients with the primary endpoint of survival, utilizing national registry data. METHODS: We queried the United Network for Organ Sharing (UNOS) data registry for all heart transplants from 1987 to 2012. We restricted our analysis to adult (≥18 yr) recipients performed from 2001-2011 (to allow for the potential for a minimum of 12 months post-transplant follow-up) who received either: no antibody based induction (NONE) or the contemporary agents (INDUCED) of either: basiliximab/daclizumab (IL-2Rab), alemtuzumab, or ATG/ALG/thymoglobulin. Kaplan-Meier estimates of the survival function as well as Cox proportional hazards models were utilized. RESULTS: Of the 17 857 heart transplants that met the inclusion criteria, there were 4635 (26%) reported deaths during the follow-up period. There were 8216 (46%) patients who were INDUCED. Of the INDUCED agents, 55% were IL-2Rab, 4% alemtuzumab, and 40% ALG/ATG/thymoglobulin. Donor and recipient characteristics were evaluated. Overall, being INDUCED did not significantly affect survival in univariable (p = 0.522) and multivariable (p = 0.130) Cox models as well as a propensity score adjusted model (p = 0.733). Among those induced, ATG/ALG/thymoglobulin appeared to have superior survival as compared with IL-2Rab (log-rank p = 0.007, univariable hazard ratio [HR] = 0.886; 95% CI: 0.811-0.968; p = 0.522). However, in a multivariable Cox model that adjusted for recipient age, VAD, BMI, steroid use, CMV match, and ischemic time, the hazard ratio for ALG/ATG/thymoglobulin vs. IL-2Rab was no longer statistically significant (HR = 0.948; 95% CI: 0.850-1.058; p = 0.341). CONCLUSION: In a contemporary analysis of heart transplant recipients, an overall analysis of induction agents does not appear to impact survival, as compared to no induction immunosuppression. While ALG/ATG/thymoglobulin appeared to have a beneficial effect on survival compared to IL-2Rab in the univariable model, this difference was no longer statistically significant once we adjusted for clinically relevant covariates.

ER stress and autophagy: new discoveries in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol.

Cyclin dependent kinase (CDK) inhibitors, such as flavopiridol, demonstrate significant single-agent activity in chronic lymphocytic leukemia (CLL), but the mechanism of action in these nonproliferating cells is unclear. Here we demonstrate that CLL cells undergo autophagy after treatment with therapeutic agents, including fludarabine, CAL-101, and flavopiridol as well as the endoplasmic reticulum (ER) stress-inducing agent thapsigargin. The addition of chloroquine or siRNA against autophagy components enhanced the cytotoxic effects of flavopiridol and thapsigargin, but not the other agents. Similar to thapsigargin, flavopiridol robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases. Both autophagy and ER stress were documented in tumor cells from CLL patients receiving flavopiridol. Thus, CLL cells undergo autophagy after multiple stimuli, including therapeutic agents, but only with ER stress mediators and CDK inhibitors is autophagy a mechanism of resistance to cell death. These findings collectively demonstrate, for the first time, a novel mechanism of action (ER stress) and drug resistance (autophagy) for CDK inhibitors, such as flavopiridol in CLL, and provide avenues for new therapeutic combination approaches in this disease.