Publications by authors named "Esteban Braggio"

93 Publications

Preneoplastic Alterations Define CLL DNA Methylome and Persist through Disease Progression and Therapy.

Blood Cancer Discov 2021 Jan 3;2(1):54-69. Epub 2020 Dec 3.

Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin 14195, Germany.

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in pre-neoplastic monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significantly differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and post-therapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state, and a distinct expression profile together with MBL cells compared to normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically-driven growth dynamics and together with its persistent presence suggests a central role in the normal-to-cancer transition.
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http://dx.doi.org/10.1158/2643-3230.BCD-19-0058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888194PMC
January 2021

Natural History of Monoclonal B-cell Lymphocytosis (MBL) Among Relatives in Chronic Lymphocytic Leukemia (CLL) Families.

Blood 2020 Dec 21. Epub 2020 Dec 21.

NCI, Bethesda, Maryland, United States.

CLL has one of the highest familial-risks among cancers. MBL, the precursor to CLL, has a higher prevalence (13-18%) in families with two or more members with CLL compared to the general population (5-12%). Although, the rate of progression to CLL for high-count MBLs (clonal B-cell count³500/µL) is ~1-5%/year, no low-count MBLs has been reported to progress to date. We report the incidence and natural history of MBL in relatives from CLL families. In 310 CLL families, we screened 1045 relatives for MBL using highly-sensitive flow cytometry and prospectively followed 449 of them. MBL incidence was directly age- and sex-adjusted to the 2010 United States population. CLL cumulative incidence was estimated using Kaplan-Meier survival curves. At baseline, the prevalence of MBL was 22% (235/1045 relatives). After a median follow-up of 8.1 years among 449 relatives, twelve individuals progressed to CLL with a 5-year cumulative incidence of 1.8%. When considering just the 139 relatives with low-count MBL, the 5-year cumulative incidence increased to 5.7%. Finally, 264 had no MBL at baseline of whom 60 individuals subsequently developed MBL (two high-count and 58 low-count MBLs) with an age- and sex-adjusted incidence of 3.5% after a median of 6 years of follow-up. In a screening cohort of relatives from CLL families, we reported progression from normal to low-count MBL to high-count MBL to CLL, demonstrating that low-count MBL precedes progression to CLL. We estimated a 1.1% annual rate of progression from low-count MBL, which is in excess to that in the general population.
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http://dx.doi.org/10.1182/blood.2020006322DOI Listing
December 2020

ROR1 targeting with the antibody drug-conjugate VLS-101 is effective in Richter syndrome patient-derived xenograft mouse models.

Blood 2021 Jan 14. Epub 2021 Jan 14.

University of Torino, Turin, Indiana, Italy.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on normal adult tissues, making it an attractive, tumor-specific therapeutic target. VLS‑101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC‑961 (a humanized immunoglobulin IgG1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate (mc-vc-PAB) linker, and the anti‑microtubule cytotoxin monomethyl auristatin E (MMAE). VLS‑101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS‑PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the post-therapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A Phase 1 clinical trial of VLS‑101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.
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http://dx.doi.org/10.1182/blood.2020008404DOI Listing
January 2021

Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma.

Nat Cancer 2020 May 27;1(5):493-506. Epub 2020 Apr 27.

Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, 02215, USA.

Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient progression. We find NK cell abundance is frequently increased in early stages, and associated with altered chemokine receptor expression. As early as SMM, we show loss of GrK memory cytotoxic T-cells, and show their critical role in MM immunosurveillance in mouse models. Finally, we report MHC class II dysregulation in CD14 monocytes, which results in T cell suppression . These results provide a comprehensive map of immune changes at play over the evolution of pre-malignant MM, which will help develop strategies for immune-based patient stratification.
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http://dx.doi.org/10.1038/s43018-020-0053-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785110PMC
May 2020

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Mol Ther 2021 Jan 1. Epub 2021 Jan 1.

T Cell Engineering, Mayo Clinic, Rochester, MN, USA; Division of Hematology, Mayo Clinic, Rochester, MN 55905, USA; Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN, USA; Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA; Department of Immunology, Mayo Clinic, Rochester, MN, USA. Electronic address:

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1 CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.
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http://dx.doi.org/10.1016/j.ymthe.2020.12.033DOI Listing
January 2021

Monosomic loss of MIR15A/MIR16-1 is a driver of multiple myeloma proliferation and disease progression.

Blood Cancer Discov 2020 Jul;1(1):68-81

Division of Hematology and Medical Oncology, Mayo Clinic, Scottsdale, AZ.

The most common genetic abnormality in multiple myeloma (MM) is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including has been mapped, the deletions in MM predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include and , but while biallelic deletion of is associated with disease progression, is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of MM spontaneously acquires del(14), syntenic to human chromosome 13, and complete inactivation, but not mutations. Taking advantage of this model, we explored the role in MM initiation and progression of two candidate loci on chromosome 13: . Monoallelic deletion of but not was sufficient to accelerate the development of monoclonal gammopathy in wildtype mice, and the progression of MM in Vk*MYC mice, resulting in increased expression of target genes and plasma cell proliferation, which was similarly observed in patients with MM.
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http://dx.doi.org/10.1158/0008-5472.bcd-19-0068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494232PMC
July 2020

Disease Flare During Temporary Interruption of Ibrutinib Therapy in Patients with Chronic Lymphocytic Leukemia.

Oncologist 2020 11 20;25(11):974-980. Epub 2020 Sep 20.

Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA.

Background: Approximately 25% of patients with chronic lymphocytic leukemia (CLL) experience a flare of disease following ibrutinib discontinuation. A critical question is whether this phenomenon may also occur when ibrutinib is temporarily held. This study aimed to determine the frequency and characteristics of disease flares in this setting and assess risk factors and clinical outcomes.

Materials And Methods: We identified all patients with CLL seen at Mayo Clinic between October 2012 and March 2019 who received ibrutinib. Temporary interruptions in treatment and associated clinical findings were ascertained.

Results: Among the 372 patients identified, 143 (38%) had at least one temporary interruption (median 1 hold, range 1-7 holds) in treatment. The median duration of interruption was 8 days (range 1-59 days) and the most common indication was periprocedural. Among the 143 patients with ≥1 hold, an associated disease flare was seen in 35 (25%) patients: mild (constitutional symptoms only) in 21 patients and severe (constitutional symptoms with exam/radiographic findings or laboratory changes) in 14 patients. Disease flare resolved with resuming ibrutinib in all patients. Predictive factors of disease flare included progressive disease at time of hold and ≥ 24 months of ibrutinib exposure. The occurrence of disease flare with an ibrutinib hold was associated with shorter event-free survival (hazard ratio 2.3; 95% confidence interval 1.3-4.1; p = .007) but not overall survival.

Conclusion: Temporary interruptions in ibrutinib treatment of patients with CLL are common, and one quarter of patients who held ibrutinib in this study experienced a disease flare. Resolution with resuming ibrutinib underscores the importance of awareness of this phenomenon for optimal management.

Implications For Practice: Ibrutinib is a very effective treatment for chronic lymphocytic leukemia (CLL) but needs to be taken continuously. Side effects, such as increased bleeding risk with procedures, require temporary interruptions in this continuous treatment. Rapid CLL progression following ibrutinib discontinuation has been increasingly recognized. This study demonstrates that similar flares in disease signs or symptoms may occur during ibrutinib holds as well. Importantly, management with restarting ibrutinib led to quick clinical improvement. Awareness of this phenomenon among clinicians is critical to avoid associated patient morbidity and premature cessation of effective treatment with ibrutinib if the flare is misidentified as true progression of disease.
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http://dx.doi.org/10.1634/theoncologist.2020-0388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648348PMC
November 2020

Chronic lymphocytic leukemia (CLL) risk is mediated by multiple enhancer variants within CLL risk loci.

Hum Mol Genet 2020 Sep;29(16):2761-2774

Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. It has a strong genetic basis, showing a ~ 8-fold increased risk of CLL in first-degree relatives. Genome-wide association studies (GWAS) have identified 41 risk variants across 41 loci. However, for a majority of the loci, the functional variants and the mechanisms underlying their causal roles remain undefined. Here, we examined the genetic and epigenetic features associated with 12 index variants, along with any correlated (r2 ≥ 0.5) variants, at the CLL risk loci located outside of gene promoters. Based on publicly available ChIP-seq and chromatin accessibility data as well as our own ChIP-seq data from CLL patients, we identified six candidate functional variants at six loci and at least two candidate functional variants at each of the remaining six loci. The functional variants are predominantly located within enhancers or super-enhancers, including bi-directionally transcribed enhancers, which are often restricted to immune cell types. Furthermore, we found that, at 78% of the functional variants, the alternative alleles altered the transcription factor binding motifs or histone modifications, indicating the involvement of these variants in the change of local chromatin state. Finally, the enhancers carrying functional variants physically interacted with genes enriched in the type I interferon signaling pathway, apoptosis, or TP53 network that are known to play key roles in CLL. These results support the regulatory roles for inherited noncoding variants in the pathogenesis of CLL.
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http://dx.doi.org/10.1093/hmg/ddaa165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530532PMC
September 2020

Proteasome Subunits Differentially Control Myeloma Cell Viability and Proteasome Inhibitor Sensitivity.

Mol Cancer Res 2020 10 19;18(10):1453-1464. Epub 2020 Jun 19.

Department of Hematology, Mayo Clinic in Arizona, Scottsdale, Arizona.

We generated eight multiple myeloma cell lines resistant to bortezomib; five acquired mutations. In 1,500 patients such mutations were rare clinically. To better understand disruption of proteasomes on multiple myeloma viability and drug sensitivity, we systematically deleted the major proteasome catalytic subunits. Multiple myeloma cells without PSMB5 were viable. Drug-resistant, PSMB5-mutated cell lines were resensitized to bortezomib by PSMB5 deletion, implying PSMB5 mutation is activating in its drug resistance function. In contrast, PSMB6 knockout was lethal to multiple myeloma cell lines. Depleting PSMB6 prevented splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8, and PSMB10; however, PSMB6 engineered without splicing function or catalytic activity, also restored viability, inferring the contribution of PSMB6 to proteasome structure to be more important than functional activity. Supporting this, bortezomib sensitivity was restored in drug-resistant multiple myeloma cell lines by low level expression of mutated PSMB6 lacking splicing function. Loss of PSMB8 and PSMB9 was neither lethal nor restored bortezomib sensitivity. Significant codependency of PSMB5, PSMB6, and PSMB7 expression was observed. We demonstrated elevated levels of PSMB6 and 7, but not 8 and 9, in some, but not all, serial patient samples exposed to proteasome inhibitors. In summary, we show PSMB6 and PSMB7, but not PSMB5, to be essential for multiple myeloma cell survival, this dependency is structural and that upregulation or activating mutation of PSMB5, 6, and 7 confers proteasome inhibitor resistance, while depletion confers sensitivity. IMPLICATIONS: These findings support modulation of PSMB5, PSMB6, or PSMB7 expression as a new therapeutic strategy.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-1026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541608PMC
October 2020

Evaluation of NUC-1031: a first-in-class ProTide in biliary tract cancer.

Cancer Chemother Pharmacol 2020 06 21;85(6):1063-1078. Epub 2020 May 21.

Division of Hematology/Oncology, Department of Internal Medicine, Mayo Clinic, Scottsdale, AZ, USA.

Purpose: NUC1031 is a first-in-class ProTide, that is a gemcitabine pro-drug designed to overcome putative mechanisms of resistance, including decreased expression of hENT/hCNT transporters, absence of activating enzymes such as deoxycytidine kinase (dCK) and presence of degrading enzymes such as cytidine deaminase (CDA). We undertook comprehensive pre-clinical evaluation of NUC1031 in biliary tract cancer (BTC) models, given that gemcitabine/cisplatin is a standard first-line therapy in advanced BTC.

Methods: Here, we compared the in vitro activity of NUC1031 in comparison to gemcitabine, validate putative mechanism(s) of action, assessed potential biomarkers of sensitivity or resistance, and performed combination studies with cisplatin. We also evaluated the in vivo efficacy of NUC1031 and gemcitabine using a CDA-high cholangiocarcinoma patient-derived xenograft (PDX) model.

Results: In a panel of BTC cell lines (N = 10), NUC1031 had less potency than gemcitabine in multiple cellular assays. NUC1031 did not demonstrate evidence of greater synergy over gemcitabine in combination with cisplatin. Surprisingly, efficacy of both gemcitabine and NUC1031 was not found to be correlated with hENT/hCTN, dCK or CDA transcript levels. Gemcitabine and NUC1031 showed equivalent efficacy in a CDA-high PDX model in vivo contradicting the primary rationale of NUC1031 design.

Conclusion: NUC1031 did not exhibit evidence of superior activity over gemcitabine, as a single-agent, or in combination with cisplatin, in either our in vivo or in vitro BTC models. Given that the largest Phase 3 study (ClinicalTrials.gov: NCT0314666) to date in BTC is underway (N = 828) comparing NUC1031/cisplatin to gemcitabine/cisplatin, our results suggest that a more conservative clinical evaluation path would be more appropriate.
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http://dx.doi.org/10.1007/s00280-020-04079-zDOI Listing
June 2020

"Direct to Drug" screening as a precision medicine tool in multiple myeloma.

Blood Cancer J 2020 05 11;10(5):54. Epub 2020 May 11.

Department of Hematology/Oncology, Mayo Clinic, Scottsdale, AZ, USA.

Seventy-six FDA-approved oncology drugs and emerging therapeutics were evaluated in 25 multiple myeloma (MM) and 15 non-Hodgkin's lymphoma cell lines and in 113 primary MM samples. Ex vivo drug sensitivities were mined for associations with clinical phenotype, cytogenetic, genetic mutation, and transcriptional profiles. In primary MM samples, proteasome inhibitors, dinaciclib, selinexor, venetoclax, auranofin, and histone deacetylating agents had the broadest cytotoxicity. Of interest, newly diagnosed patient samples were globally less sensitive especially to bromodomain inhibitors, inhibitors of receptor tyrosine kinases or non-receptor kinases, and DNA synthesis inhibitors. Clustering demonstrated six broad groupings of drug sensitivity linked with genomic biomarkers and clinical outcomes. For example, our findings mimic clinical observations of increased venetoclax responsiveness in t(11;14) patients but also identify an increased sensitivity profile in untreated patients, standard genetic risk, low plasma cell S-Phase, and in the absence of Gain(1q) and t(4;14). In contrast, increased ex vivo responsiveness to selinexor was associated with biomarkers of poor prognosis and later relapse patients. This "direct to drug" screening resource, paired with functional genomics, has the potential to successfully direct appropriate individualized therapeutic approaches in MM and to enrich clinical trials for likely responders.
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http://dx.doi.org/10.1038/s41408-020-0320-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214452PMC
May 2020

Tumor mutational load predicts time to first treatment in chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis beyond the CLL international prognostic index.

Am J Hematol 2020 08 7;95(8):906-917. Epub 2020 May 7.

Division of Hematology /Oncology, Mayo Clinic, Scottsdale, Arizona, USA.

Next-generation sequencing identified about 60 genes recurrently mutated in chronic lymphocytic leukemia (CLL). We examined the additive prognostic value of the total number of recurrently mutated CLL genes (i.e., tumor mutational load [TML]) or the individually mutated genes beyond the CLL international prognostic index (CLL-IPI) in newly diagnosed CLL and high-count monoclonal B-cell lymphocytosis (HC MBL). We sequenced 59 genes among 557 individuals (112 HC MBL/445 CLL) in a multi-stage design, to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time-to-first treatment (TTT), adjusted for CLL-IPI and sex. TML was associated with shorter TTT in the discovery and validation cohorts, with a combined estimate of continuous HR = 1.27 (CI:1.17-1.39, P = 2.6 × 10 ; c-statistic = 0.76). When stratified by CLL-IPI, the association of TML with TTT was stronger and validated within low/intermediate risk (combined HR = 1.54, CI:1.37-1.72, P = 7.0 × 10 ). Overall, 80% of low/intermediate CLL-IPI cases with two or more mutated genes progressed to require therapy within 5 years, compared to 24% among those without mutations. TML was also associated with shorter TTT in the HC MBL cohort (HR = 1.53, CI:1.12-2.07, P = .007; c-statistic = 0.71). TML is a strong prognostic factor for TTT independent of CLL-IPI, especially among low/intermediate CLL-IPI risk, and a better predictor than any single gene. Mutational screening at early stages may improve risk stratification and better predict TTT.
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http://dx.doi.org/10.1002/ajh.25831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409825PMC
August 2020

Mate pair sequencing outperforms fluorescence in situ hybridization in the genomic characterization of multiple myeloma.

Blood Cancer J 2019 12 16;9(12):103. Epub 2019 Dec 16.

Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.

Fluorescence in situ hybridization (FISH) is currently the gold-standard assay to detect recurrent genomic abnormalities of prognostic significance in multiple myeloma (MM). Since most translocations in MM involve a position effect with heterogeneous breakpoints, we hypothesize that FISH has the potential to miss translocations involving these regions. We evaluated 70 bone marrow samples from patients with plasma cell dyscrasia by FISH and whole-genome mate-pair sequencing (MPseq). Thirty cases (42.9%) displayed at least one instance of discordance between FISH and MPseq for each primary and secondary abnormality evaluated. Nine cases had abnormalities detected by FISH that went undetected by MPseq including 6 tetraploid clones and three cases with missed copy number abnormalities. In contrast, 19 cases had abnormalities detected by MPseq that went undetected by FISH. Seventeen were MYC rearrangements and two were 17p deletions. MPseq identified 36 MYC abnormalities and 17 (50.0% of MYC abnormal group with FISH results) displayed a false negative FISH result. MPseq identified 10 cases (14.3%) with IgL rearrangements, a recent marker of poor outcome, and 10% with abnormalities in genes associated with lenalidomide response or resistance. In summary, MPseq was superior in the characterization of rearrangement complexity and identification of secondary abnormalities demonstrating increased clinical value compared to FISH.
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http://dx.doi.org/10.1038/s41408-019-0255-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914798PMC
December 2019

Identification of PIKfyve kinase as a target in multiple myeloma.

Haematologica 2020 Jun 3;105(6):1641-1649. Epub 2019 Oct 3.

Division of Hematology/Oncology, Mayo Clinic Arizona, Scottsdale, AZ

The cellular cytotoxicity of APY0201, a PIKfyve inhibitor, against multiple myeloma was initially identified in an unbiased chemical library screen. The activity of APY0201 was confirmed in all 25 cell lines tested and in 40% of 100 patient-derived primary samples, with increased activity in primary samples harboring trisomies and lacking t(11;14). The broad anti-multiple myeloma activity of PIKfyve inhibitors was further demonstrated in confirmatory screens and showed the superior potency of APY0201 when compared to the PIKfyve inhibitors YM201636 and apilimod, with a mid-point half maximal effective concentration (EC) at nanomolar concentrations in, respectively, 65%, 40%, and 5% of the tested cell lines. Upregulation of genes in the lysosomal pathway and increased cellular vacuolization were observed following APY0201 treatment, although these cellular effects did not correlate well with responsiveness. We confirm that PIKfyve inhibition is associated with activation of the transcription factor EB, a master regulator of lysosomal biogenesis and autophagy. Furthermore, we established an assay measuring autophagy as a predictive marker of APY0201 sensitivity. Overall, these findings indicate promising activity of PIKfyve inhibitors secondary to disruption of autophagy in multiple myeloma and suggest a strategy to enrich for likely responders.
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http://dx.doi.org/10.3324/haematol.2019.222729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271606PMC
June 2020

Ibrutinib-Rituximab or Chemoimmunotherapy for Chronic Lymphocytic Leukemia.

N Engl J Med 2019 08;381(5):432-443

From Stanford University, Stanford (T.D.S., S.E.C.), the University of California, Irvine, Medical Center, Orange (S.O.), and Kaiser Permanente National Cancer Institute Community Oncology Research Program (NCORP)-Permanente Medical Group, Oakland (C.C.Z.) - all in California; Dana-Farber Cancer Institute, Boston (X.V.W., R.M.S.); Mayo Clinic, Rochester (N.E.K., C.A.H., J.F.L., M.L.), and Minnesota Oncology, Burnsville (A.K.S.) - both in Minnesota; Northwell Health Cancer Institute, Donald and Barbara Zucker School of Medicine at Hofstra-Northwell, Lake Success (J.B.), and University of Rochester, Rochester (P.M.B.) - both in New York; Mayo Clinic, Phoenix, AZ (D.F.J., E.B.); Washington University School of Medicine, St. Louis (A.F.C.); Memorial Sloan Kettering Cancer Center, New York (A.R.M., M.T.); Aurora Cancer Care, West Allis, WI (M.P.M.); National Cancer Institute, Bethesda, MD (R.F.L.); and the University of Alabama, Tuscaloosa (H.E.).

Background: Data regarding the efficacy of treatment with ibrutinib-rituximab, as compared with standard chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab, in patients with previously untreated chronic lymphocytic leukemia (CLL) have been limited.

Methods: In a phase 3 trial, we randomly assigned (in a 2:1 ratio) patients 70 years of age or younger with previously untreated CLL to receive either ibrutinib and rituximab for six cycles (after a single cycle of ibrutinib alone), followed by ibrutinib until disease progression, or six cycles of chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab. The primary end point was progression-free survival, and overall survival was a secondary end point. We report the results of a planned interim analysis.

Results: A total of 529 patients underwent randomization (354 patients to the ibrutinib-rituximab group, and 175 to the chemoimmunotherapy group). At a median follow-up of 33.6 months, the results of the analysis of progression-free survival favored ibrutinib-rituximab over chemoimmunotherapy (89.4% vs. 72.9% at 3 years; hazard ratio for progression or death, 0.35; 95% confidence interval [CI], 0.22 to 0.56; P<0.001), and the results met the protocol-defined efficacy threshold for the interim analysis. The results of the analysis of overall survival also favored ibrutinib-rituximab over chemoimmunotherapy (98.8% vs. 91.5% at 3 years; hazard ratio for death, 0.17; 95% CI, 0.05 to 0.54; P<0.001). In a subgroup analysis involving patients without immunoglobulin heavy-chain variable region () mutation, ibrutinib-rituximab resulted in better progression-free survival than chemoimmunotherapy (90.7% vs. 62.5% at 3 years; hazard ratio for progression or death, 0.26; 95% CI, 0.14 to 0.50). The 3-year progression-free survival among patients with mutation was 87.7% in the ibrutinib-rituximab group and 88.0% in the chemoimmunotherapy group (hazard ratio for progression or death, 0.44; 95% CI, 0.14 to 1.36). The incidence of adverse events of grade 3 or higher (regardless of attribution) was similar in the two groups (in 282 of 352 patients [80.1%] who received ibrutinib-rituximab and in 126 of 158 [79.7%] who received chemoimmunotherapy), whereas infectious complications of grade 3 or higher were less common with ibrutinib-rituximab than with chemoimmunotherapy (in 37 patients [10.5%] vs. 32 [20.3%], P<0.001).

Conclusions: The ibrutinib-rituximab regimen resulted in progression-free survival and overall survival that were superior to those with a standard chemoimmunotherapy regimen among patients 70 years of age or younger with previously untreated CLL. (Funded by the National Cancer Institute and Pharmacyclics; E1912 ClinicalTrials.gov number, NCT02048813.).
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http://dx.doi.org/10.1056/NEJMoa1817073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908306PMC
August 2019

Identification of lenalidomide resistance pathways in myeloma and targeted resensitization using cereblon replacement, inhibition of STAT3 or targeting of IRF4.

Blood Cancer J 2019 02 11;9(2):19. Epub 2019 Feb 11.

Division of Hematology, Mayo Clinic, Scottsdale, AZ, USA.

To understand immunomodulatory drug (IMiD) resistance in multiple myeloma (MM), we created isogenic human multiple myeloma cell lines (HMCLs) sensitive and resistant to lenalidomide, respectively. Four HMCLs were demonstrated to be resistant to all IMiDs including lenalidomide, pomalidomide, and CC-220, but not to Bortezomib. In three HMLCs (MM.1.SLenRes, KMS11LenRes and OPM2LenRes), CRBN abnormalities were found, including chromosomal deletion, point mutation, and low CRBN expression. The remaining HMCL, XG1LenRes, showed no changes in CRBN but exhibited CD147 upregulation and impaired IRF4 downregulation after lenalidomide treatment. Depletion of CD147 in XG1LenRes and three additional HMCLs had no significant impact on MM viability and lenalidomide response. Further analysis of XG1LenRes demonstrated increased IL6 expression and constitutive STAT3 activation. Inhibition of STAT3 with a selective compound (PB-1-102) re-sensitized XG1LenRes to lenalidomide. Since XG1LenRes harbors a truncated IRF4 that is not downregulated by lenalidomide, we targeted IRF4/MYC axis with a selective inhibitor of the bromodomain of CBP/EP300 (SGC-CBP30), which restored lenalidomide response in XG1LenRes. This strategy also appeared to be more broadly applicable as SGC-CBP30 could re-sensitize two resistant HMCLs with low but detectable CRBN expression to lenalidomide, suggesting that targeting CBP/E300 is a promising approach to restore IMiD sensitivity in MM with detectable CRBN expression.
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http://dx.doi.org/10.1038/s41408-019-0173-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370766PMC
February 2019

IGH translocations in chronic lymphocytic leukemia: Clinicopathologic features and clinical outcomes.

Am J Hematol 2019 03 8;94(3):338-345. Epub 2019 Jan 8.

Division of Hematology, Mayo Clinic, Rochester, Minnesota.

The prevalence, clinicopathologic correlates, and outcomes of previously untreated chronic lymphocytic leukemia (CLL) patients with IGH-BCL2 and IGH-BCL3 translocations are not well known. Using the Mayo Clinic CLL database, we identified patients seen between March 1, 2002 and September 30, 2016 who had FISH testing performed within 3 years of CLL diagnosis. The prognostic profile, time to first therapy (TTT), and overall survival (OS) of patients with IGH-BCL2 and IGH-BCL3 translocation were compared to patients without these abnormalities (non-IGH group). Of 1684 patients who met the inclusion criteria, 38 (2.2%) had IGH-BCL2, and 16 (0.9%) had IGH-BCL3 translocation at diagnosis. Patients with IGH-BCL3 translocation were more likely to have high and very-high CLL-International Prognostic Index, compared to patients with IGH-BCL2 translocation and the non-IGH group. The 5-year probability of requiring therapy was significantly higher for IGH-BCL3 compared to IGH-BCL2 and non-IGH groups (84% vs 33% vs 29%, respectively, P < 0.0001). The 5-year OS was significantly shorter for IGH-BCL3 compared to IGH-BCL2 and non-IGH groups (45% vs 89% vs 86%, respectively, P < 0.0001). On multivariable analyses, IGH-BCL3 translocation was associated with a shorter TTT (hazard ratio [HR] = 2.7; P = 0.005) and shorter OS (HR = 5.5; P < 0.0001); IGH-BCL2 translocation did not impact TTT and OS. In conclusion, approximately 3% of all newly diagnosed CLL patients have either an IGH-BCL2 or IGH-BCL3 translocation. Patients with IGH-BCL3 translocations have a distinct prognostic profile and outcome. These results support the inclusion of an IGH probe during the routine evaluation of FISH abnormalities in newly diagnosed CLL.
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http://dx.doi.org/10.1002/ajh.25385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625355PMC
March 2019

Loss of TNFAIP3 enhances MYD88-driven signaling in non-Hodgkin lymphoma.

Blood Cancer J 2018 10 9;8(10):97. Epub 2018 Oct 9.

Division of Hematology, Mayo Clinic, Rochester, MN, USA.

MYD88 mutations are one of the most recurrent mutations in hematologic malignancies. However, recent mouse models suggest that MYD88 alone may not be sufficient to induce tumor formation. Interplay between MYD88 and other genetic events is further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88. However, we are still lacking information about the consequence of MYD88 in combination with TNFAIP3 loss in human B cell lymphoma. Review of our genetic data on diffuse large B cell lymphoma (DLBCL) and Waldenstrom macroglobulinemia (WM), found that a large percentage of DLBCL and WM cases that have a MYD88 mutation also harbor a TNFAIP3 loss, 55% DLBCL and 28% of WM, respectively. To mimic this combination of genetic events, we used genomic editing technology to knock out TNFAIP3 in MYD88 non-Hodgkin's lymphoma (NHL) cell lines. Loss of A20 expression resulted in increased NF-κB and p38 activity leading to upregulation of the NF-κB target genes BCL2 and MYC. Furthermore, we detected the increased production of IL-6 and CXCL10 which led to an upregulation of the JAK/STAT pathway. Overall, these results suggest that MYD88 signaling can be enhanced by a second genetic alteration in TNFAIP3 and highlights a potential opportunity for therapeutic targeting.
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http://dx.doi.org/10.1038/s41408-018-0130-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177394PMC
October 2018

Spectrum and functional validation of PSMB5 mutations in multiple myeloma.

Leukemia 2019 02 19;33(2):447-456. Epub 2018 Jul 19.

Department of Hematology-Oncology, Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.

Despite an increasing number of approved therapies, multiple myeloma (MM) remains an incurable disease and only a small number of patients achieve prolonged disease control. Some genes have been linked with response to commonly used anti-MM compounds, including immunomodulators (IMiDs) and proteasome inhibitors (PIs). In this manuscript, we demonstrate an increased incidence of acquired proteasomal subunit mutations in relapsed MM compared to newly diagnosed disease, underpinning a potential role of point mutations in the clonal evolution of MM. Furthermore, we are first to present and functionally characterize four somatic PSMB5 mutations from primary MM cells identified in a patient under prolonged proteasome inhibition, with three of them affecting the PI-binding pocket S1. We confirm resistance induction through missense mutations not only to Bortezomib, but also, in variable extent, to the next-generation PIs Carfilzomib and Ixazomib. In addition, a negative impact on the proteasome activity is assessed, providing a potential explanation for later therapy-induced eradication of the affected tumor subclones in this patient.
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http://dx.doi.org/10.1038/s41375-018-0216-8DOI Listing
February 2019

Protocol for MP: A Comprehensive and Clinical Oriented Targeted Sequencing Panel for Routine Molecular Analysis in Multiple Myeloma.

Methods Mol Biol 2018 ;1792:117-128

Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.

Over the past 10 years next generation sequencing (NGS) approaches deciphered a large number of genomes from a wide variety of tumor types. However, despite most relevant findings, this technology has not yet been implemented into standard diagnostic workflows. Broad access to NGS technology is still limited, sequencing/analysis times exceed clinically relevant timeframes and despite huge cuts, costs remain significant. We proposed a custom-tailored gene panel, which focuses on a selected number of relevant genes and developed a clinically oriented NGS targeted sequencing approach for the molecular characterization of Multiple Myeloma (MM) tumors, allowing the description of the tumor genetic heterogeneity and its changes under selective pressure of antitumor therapy, in a more cost effective and faster turnaround timeframe.
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http://dx.doi.org/10.1007/978-1-4939-7865-6_8DOI Listing
February 2019

Novel Richter Syndrome Xenograft Models to Study Genetic Architecture, Biology, and Therapy Responses.

Cancer Res 2018 07 7;78(13):3413-3420. Epub 2018 May 7.

Department of Medical Sciences, University of Turin, Italy.

Richter syndrome represents the evolution of chronic lymphocytic leukemia into an aggressive tumor, most commonly diffuse large B-cell lymphoma. The lack of and models has severely hampered drug testing in a disease that is poorly responsive to common chemoimmunotherapeutic combinations as well as to novel kinase inhibitors. Here we report for the first time the establishment and genomic characterization of two patient-derived tumor xenograft (PDX) models of Richter syndrome, RS9737 and RS1316. Richter syndrome xenografts were genetically, morphologically, and phenotypically stable and similar to the corresponding primary tumor. RS9737 was characterized by biallelic inactivation of and monoallelic deletion of 11q23 (), and mutations of , and RS1316 carried trisomy 12 and showed mutations in , and RNA sequencing confirmed that in both cases >80% of the transcriptome was shared between primary tumor and PDX. In line with the mutational profile, pathway analyses revealed overactivation of the B-cell receptor, NFκB, and NOTCH pathways in both tumors, potentially providing novel tumor targets. In conclusion, these two novel models of Richter syndrome represent useful tools to study biology and response to therapies of this highly aggressive and still incurable tumor. Two patient-derived xenograft models of Richter syndrome represent the first model to study biology of the disease and to test novel therapeutic strategies. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-4004DOI Listing
July 2018

Concurrent progressive multifocal leukoencephalopathy and central nervous system infiltration by multiple myeloma: A case report.

J Oncol Pharm Pract 2019 Jun 24;25(4):998-1002. Epub 2018 Apr 24.

1 Department of Hematology, Hospital Universitario 12 de Octubre, Hematological Malignancies Clinical Research Unit H120-CNIO, Madrid, Spain.

Progressive multifocal leukoencephalopathy rarely occurs in patients with multiple myeloma. Intracranial central nervous system invasion is also an uncommon event in multiple myeloma, occurring in less than 1% of cases. We describe herein an exceptional case of coexisting progressive multifocal leukoencephalopathy and intraparenchymal central nervous system myeloma infiltration. A 73-year-old woman with relapsed multiple myeloma was treated with 15 cycles of lenalidomide and dexamethasone, but therapy had to be stopped because of a hip fracture after a fall. During hospitalization, the patient developed progressive multifocal leukoencephalopathy caused by John Cunningham virus, and a prominent intra-parenchymal CD138-positive infiltrate was detected. VDJ rearrangements of the immunoglobulin heavy chain gene and the mutational profile of plasma cells in bone marrow at the time of diagnosis and in brain biopsy after progression were analyzed by next generation sequencing, showing genetic differences between medullary and extramedullary myeloma cells. The role of long-term treatment with lenalidomide and dexamethasone in the development progressive multifocal leukoencephalopathy or intraparenchymal central nervous system myeloma infiltration remains unknown. However, our results suggest that both events may have arisen as a consequence of treatment-related immunosuppression. Thus, an appropriate clinical approach compatible with the simultaneous treatment of progressive multifocal leukoencephalopathy and multiple myeloma should be developed.
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http://dx.doi.org/10.1177/1078155218769367DOI Listing
June 2019

Evaluation of Revised International Staging System (R-ISS) for transplant-eligible multiple myeloma patients.

Ann Hematol 2018 Aug 6;97(8):1453-1462. Epub 2018 Apr 6.

Division of Hematology and Oncology Mayo Clinic, Scottsdale, AZ, USA.

The International Myeloma Working Group has proposed the Revised International Staging System (R-ISS) for risk stratification of multiple myeloma (MM) patients. There are a limited number of studies that have validated this risk model in the autologous stem cell transplant (ASCT) setting. In this retrospective study, we evaluated the applicability and value for predicting survival of the R-ISS model in 134 MM patients treated with new agents and ASCT at the Mayo Clinic in Arizona and the University Hospital of Salamanca in Spain. The patients were reclassified at diagnosis according to the R-ISS: 44 patients (33%) had stage I, 75 (56%) had stage II, and 15 (11%) had stage III. After a median follow-up of 60 months, R-ISS assessed at diagnosis was an independent predictor for overall survival (OS) after ASCT, with median OS not reached, 111 and 37 months for R-ISS I, II and III, respectively (P < 0.001). We also found that patients belonging to R-ISS II and having high-risk chromosomal abnormalities (CA) had a significant shorter median OS than those with R-ISS II without CA: 70 vs. 111 months, respectively. Therefore, this study lends further support for the R-ISS as a reliable prognostic tool for estimating survival in transplant myeloma patients and suggests the importance of high-risk CA in the R-ISS II group.
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http://dx.doi.org/10.1007/s00277-018-3316-7DOI Listing
August 2018

Genetic Alterations in Essential Thrombocythemia Progression to Acute Myeloid Leukemia: A Case Series and Review of the Literature.

Front Oncol 2018 19;8:32. Epub 2018 Feb 19.

Bone Marrow Transplantation Unit, Specialized Laboratories, Laboratory of Molecular Biology, National Cancer Institute (INCa), Rio de Janeiro, Brazil.

The genetic events associated with transformation of myeloproliferative neoplasms (MPNs) to secondary acute myeloid leukemia (sAML), particularly in the subgroup of essential thrombocythemia (ET) patients, remain incompletely understood. Deep studies using high-throughput methods might lead to a better understanding of genetic landscape of ET patients who transformed to sAML. We performed array-based comparative genomic hybridization (aCGH) and whole exome sequencing (WES) to analyze paired samples from ET and sAML phases. We investigated five patients with previous history of MPN, which four had initial diagnosis of ET (one case harboring p.Val617Phe and the remaining three type II p.Lys385fs*47), and one was diagnosed with MPN/myelodysplastic syndrome with thrombocytosis ( p.Lys700Glu). All were homogeneously treated with hydroxyurea, but subsequently transformed to sAML (mean time of 6 years/median of 4 years to transformation). Two of them have chromosomal abnormalities, and both acquire 2p gain and 5q deletion at sAML stage. The molecular mechanisms associated with leukemic progression in MPN patients are not clear. Our WES data showed alterations recurrently observed as mutations (missense and frameshift) and monoallelic loss. On the other hand, aCGH showed novel chromosome abnormalities (+2p and del5q) potentially associated with disease progression. The results reported here add valuable information to the still fragmented molecular basis of ET to sAML evolution. Further studies are necessary to identify minimal deleted/amplified region and genes relevant to sAML transformation.
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http://dx.doi.org/10.3389/fonc.2018.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826070PMC
February 2018

CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma.

Mol Cancer Ther 2017 Dec 27;16(12):2862-2870. Epub 2017 Sep 27.

Department of Hematology, Mayo Clinic in Arizona, Scottsdale, Arizona.

Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796678PMC
December 2017