An exploratory clinical trial of bortezomib in patients with lower risk myelodysplastic syndromes

Abstract

Myelodysplastic syndromes (MDSs) are characterized by ineffective hematopoiesis and an increased risk of transformation. Few effective therapies are available for lower risk MDS patients, especially after the failure of hypomethylating agents. MDS progenitor cells are dependent on the nuclear factor-κB (NF-κB) for survival, which makes it an attractive therapeutic target. As a proteosomal inhibitor, bortezomib is thought to have inhibitory activity against NF-κB. We designed a proof-of-principle study of subcutaneous (SC) bortezomib in lower risk MDS patients with evidence of NF-κB activation in their bone marrow. Fifteen patients were treated, their median age was 71 (range 56–87), 33% were low and 67% int-1 by IPSS, median number of prior therapies was 2, all patients were transfusion dependent. Baseline median pp65 percentage was 31% and 11 patients had evidence of ring sideroblasts. SC bortezomib was safe, well tolerated with no excess toxicity. Three patients out of the 15 (20%) had evidence of response with hematologic improvement (HI-E). Bortezomib caused a decrease in pp65 levels in 7 out of 13 evaluable patients (54%, p=0.025). Of interest, unexpectedly, we observed a significant decrease in ring sideroblasts in 7 out of 10 (70%) evaluable patients during treatment. In conclusion, this study suggests that NF-κB activation, measured by pp65 levels, may be a useful biomarker in MDS. Bortezomib is safe in this patient population but has modest clinical activity. The role of the proteasome in the genesis of ring sideroblasts needs further study.

Introduction

Myelodysplastic syndromes (MDS) represent a spectrum of clonal hematopoietic disorders that affect the myeloid lineage, characterized by ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia (AML).[1] Patients with MDS are stratified into lower or higher-risk categories based on IPSS or IPSS-R classifications.[2, 3] For a majority of lower risk (low or int-1) MDS patients, the standard of care remains suboptimal with few effective therapeutic alternatives, particularly for patients previously treated with a hypomethylating agent.[4] Novel treatment approaches are needed for these patients.

Accumulating evidence from recent clinical and molecular studies supports a key role for deregulation of the innate immune system and inflammatory signaling in the pathogenesis of MDS.[5] Toll-like receptors (TLRs) are a family of pattern-recognition receptors that play a pivotal role in the innate immunity. These receptors activate a common signaling pathway through adaptor protein MyD88, culminating in the activation of nuclear factor-kB (NF-κB).[6, 7] Recent studies in MDS demonstrated a constitutive activation of genes that involve a variety of different steps of this pathway.[8–11] This evidence implicates NF-κB in the pathogenesis of MDS, and, hence, makes it a potential therapeutic target.

The NF-κB transcription factor family is composed of five proteins (p50, p52, p65, c-Rel, and RelB) that play a role in apoptosis regulation, cell proliferation, survival, and metastasis.[12] It is, therefore, not surprising that it has been linked to the pathogenesis of multiple solid and hematologic malignancies.[12–15] Within the canonical pathway, members of the NF-κB family form dimers (most commonly heterodimers of p65 with p50) which are retained in the cytoplasm through interactions with inhibitory molecules of the inhibitor of the NF-κB (IκB) family.[12, 16] Activating stimuli (including cytokines and various stress signals) lead to phosphorylation of the IκB molecules by IκB kinases (IKKs) and their degradation via the ubiquitin-proteasome pathway.[17] Consequently, the NF-κB dimers are liberated and are then free to translocate to the nucleus and activate their target genes.[18] Phosphorylation of p65 is required for nuclear translocation and transcriptional activation of target genes involved in proliferation, stress response and cytokine production.[19, 20] Therefore, phosphorylated p65 (pp65) may be used as a clinical surrogate for NF-κB activation.

The NF-κB subunit RelA/p65 has also been described as an important factor in hematopoiesis through the regulation of hematopoietic stem cell (HSC) function and lineage commitment. Also, p65 has been shown to play a potential role in the regulation of cell differentiation via up-regulation of additional NF-κB subunits; suggesting that, p65 may influence specific lineage-committed cell types.[21, 22]

Bortezomib is a reversible selective inhibitor of the 26S proteosomal subunit with previously described inhibitory activity against NF-κB.[23–25] In preclinical and clinical studies, bortezomib was shown to induce apoptosis in malignant cells of hematopoietic lineage via inhibition of the NF-κB pathway.[26, 27] Leukemic stem cells are highly dependent on NF-κB for survival;[28, 29] MDS progenitors are also highly dependent on NF-κB activation for survival.[10, 30] In vitro studies of MDS cells have shown that NF-κB inhibition leads to an autophagic stress response followed by apoptotic cell death.[31] In clinical studies, bortezomib has shown modest activity in myeloid neoplasms, both as a single agent and in combination with other cytotoxic agents.[32–36] Comparison between subcutaneous (SC) and intravenous administration of bortezomib showed that SC administration offers similar efficacy, with improved safety profile, especially with a significantly lower risk of peripheral neuropathy.[37, 38]

As a proof of concept, we designed this study using SC bortezomib in patients with low or intermediate-1 risk MDS who had evidence of NF-κB activation. Our objectives were primarily to determine the effect of SC bortezomib on modulation of NF-κB activity and secondarily to determine the clinical activity, safety and tolerability of SC bortezomib in these patients.

Patients and Methods

Eligibility criteria

Eligible patients had a confirmed diagnosis of MDS according to WHO or FAB criteria[39–41], and were classified by the IPSS as low or int-1 risk MDS within 28 days of the first dose of treatment. Patients were required to have a presence of the phosphorylated p65 NF-κB component in at least 5% of their bone marrow cells. Eligible patients were at least 18 years of age, had an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less, and must have received at least one prior therapy for MDS. Any prior systemic treatment had to be completed 14 days prior to the study start date, and any prior radiation therapy had to be completed within 3 weeks prior to enrollment. Patients who had received a prior allogeneic stem cell transplant for MDS were allowed to participate in the study. Patients were required to have adequate liver and renal function and were required to practice effective methods of contraception if not surgically sterile. Female patients of childbearing age were required to have a negative pregnancy test within 72 hours of treatment.

Patients were excluded if they had any severe concurrent disease or condition that would make them inappropriate for study participation, including a myocardial infarction within 6 months of enrollment, New York Heart Association (NYHA) Class III or IV heart failure, uncontrolled angina, uncontrolled ventricular arrhythmias, or electrocardiographic evidence of active ischemia or active conduction system abnormalities. Patients with other concomitant malignancies were excluded, with the exception of a completely resected basal cell carcinoma or squamous cell carcinoma of the skin, an in situ malignancy, or low-risk prostate cancer after curative therapy. Other exclusion criteria included confirmed pregnancy or lactation, hypersensitivity to bortezomib, boron or mannitol and peripheral neuropathy grade 2 or higher.

Correlative studies

We analyzed phosphorylated-p65 (pp65) level at baseline on day 21 of cycles 1 and 3 and then in subsequent marrows as clinically indicated.

Bone marrow aspirate smears were fixed for 30 minutes in fresh absolute methanol at 2–8 °C. After fixation, the thick area of the bone marrow smears was scraped off the slide while wet with methanol and allowed to air dry for 10 minutes at room temperature. Following incubation for 20 minutes in 1X TBST (Tris-Buffer Saline Tween 20, Dako Noth America inc. Carpinteria, CA), immunofluorescence was assessed by an indirect method in a Dako autostainer Link 48 at room temperature. Smears were stained using 1:20 of 300 ul of the rabbit polyclonal antibody reactive with NF-kB p65 (p-NFқB p65 (Ser276) antibody, Santa Cruz Biotechnology, TX, USA) for 30 minutes followed by 1 wash with 1X TBST. This assay was performed in the Department of Hematopathology following Clinical Laboratory Improvement Amendments (CLIA) regulations at The University of Texas MD Anderson Cancer Center. The antibody–antigen complexes were detected by incubation for 30 minutes using 1:5 of 300 ul of FITC–conjugated secondary goat anti-rabbit immunoglobulin G, (Fab2) antibody (FITC, Supertechs inc. MD, USA) and 2 washings of 1X TBST. Slides were embedded in antifade Vectashield mounting medium with Propidium iodide (PI); (Vectashield mounting medium with PI, Vectashield, CA, USA) cover slipped, and analyzed with a conventional fluorescence microscope Olympus BX53 and DP73 digital camera with CellSens program (Olympus BX53 and DP73 digital camera with CellSens program, Olympus America inc. MA, USA).[42] Positive results were defined as nuclear fluorescence phosphorylated p65 NF-κB in at least 5% of bone marrow cells.

Cytogenetic studies

Conventional chromosomal analysis was performed on G-banded metaphases that were prepared from unstimulated 24-hour and 48-hour BM aspirate cultures using standard techniques. The results were reported using the 2016 International System for Human Cytogenetics Nomenclature as described previously.[43, 44]

Molecular studies

We performed mutation analysis using a 28-gene panel as previously described. [45–48] Briefly, genomic DNA was extracted from bone marrow aspirates or peripheral blood. Amplicon-based next-generation sequencing (NGS) targeting the entire coding regions of a panel of 28 genes associated with myeloid neoplasms was performed using a MiSeq platform (Illumina, San Diego, CA). The genes analyzed included ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, and WT1. A sequencing library was prepared using 250 ng of DNA template. Equal quantities of DNA from purified sequencing libraries were used for TruSeq paired-end sequencing on the MiSeq sequencer using the MiSeq Reagent Kit v2 (500 cycles). Variant calling was performed with Illumina MiSeq Reporter Software using human genome build 19 (hg 19) as a reference. For clinical reporting, a minimum sequencing coverage of 250X (bi-directional true paired-end sequencing) was required. The analytical sensitivity was established at 5% mutant reads in a background of wild-type reads. CEBPA was performed using PCR followed by Sanger sequencing. Internal tandem duplications (ITD) of FLT3 gene and NPM1 mutation (exon12) were assessed using PCR followed by capillary electrophoresis (all of these methods have been previously described).[45, 49, 50] PCR-based DNA sequencing was then used to identify: RAS mutation (codons 12, 13 and 61 of the KRAS and NRAS), IDH1R132 mutation (codon 87 to138 in exon 4), IDH2 mutation (exon 4) and c-KIT mutation (exon 17) as previously described.[51]

Study design and treatment plan

This was a single arm proof of concept study designed primarily to determine the effect of SC bortezomib on modulation of NF-κb activity, and secondarily to determine the clinical activity and safety and tolerability of SC bortezomib in patients with low or intermediate-1 risk MDS who had evidence of NF-κB activation.

This study was approved by the University of Texas MD Anderson Cancer Center’s Institutional Review Board in accordance with an assurance filed with and approved by the Department of Health and Human Services. Informed written consent was obtained from each patient before enrollment in the study. This trial is registered at www.clinicaltrials.gov as NCT01891968.

Bortezomib was administered via subcutaneous route at a dose of 1.3 mg/m² on days 1, 4, 8 and 11 of each cycle. The duration of each treatment cycle was 21 days. Patients received the first cycle of therapy without interruption regardless of the degree of myelosuppression. After the first course of therapy, the interval between cycles of therapy could be spaced out at the discretion of the treating physician. The dose and schedule were adapted based on PK and safety data for SC bortezomib as published by Moreau et al.[37] Doses were held and sequentially reduced for treatment-related adverse events, as defined in the protocol. The first dose reduction level was 1mg/m² and the second dose reduction level was 0.7 mg/m². If toxicity occurred after the second dose reduction, the treatment was discontinued; dose re-escalation was not permitted.

Response and toxicity assessment

Adverse events were graded and reported according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. All patients were considered evaluable for toxicity from the time of their first treatment with bortezomib. All treated patients were evaluated for response. Response criteria were assessed according to the modified IWG criteria.[52, 53] Responses included complete remission (defined as bone marrow ≤ 5% myeloblasts with normal maturation of all cell lines and peripheral blood Hgb 11g/dL, platelets 100×10⁹/L, neutrophils 1.0×10⁹/L, blasts 0%), partial remission (defined as bone marrow blasts decreased by 50% over pretreatment but still > 5%), marrow complete remission (defined as bone marrow ≤ 5% myeloblasts and decrease by 50% over pretreatment) and any hematological improvement achieved at any time during the duration of the therapy. Event-free survival (EFS) was defined as the time between the start of therapy and the date of lack of response, loss of response, transformation to acute myeloid leukemia, or death, whichever occurred first. OS was defined as the time between the start of therapy and death. Patients who were alive at the last follow-up date were censored in survival analyses.The protocol is available in the supplementary material section for detailed definitions of response criteria.

Statistical considerations

The primary objective of the study was to investigate the effect of SC bortezomib on the modulation of NF-κb activity by pp65 immunofluorescence staining in patients with low or intermediate-1 MDS. The secondary objectives were to determine the clinical activity, safety and tolerability of SC bortezomib in patients with low or intermediate-1 MDS. The primary efficacy outcome was the overall response rate based mainly on hematologic improvement as defined by the IWG, but could include complete remission, partial remission and marrow complete remission. A maximum of 40 evaluable patients could be enrolled, but the study would be subject to premature discontinuation if the data suggested that: Pr(θ <0.15|data)>0.95 where θ was the overall response (OR) rate. Currently the standard practice in low risk MDS is observation; therefore, an OR rate of about 15% was worth considering. We assumed the OR rate had a prior Beta distribution (0.3, 1.7) with mean of 0.15 and variance of 0.0425. The study was to be discontinued prematurely if at any time we determined that there was a greater than 95% chance that the average OR rate was less than 15%. The proportion of patients having hematologic improvement and the proportion of patients having grade 3 or higher toxicities were estimated by a Bayesian posterior credible interval. Survival probabilities were calculated by using the Kaplan-Meier method and were assessed from the time of therapy initiation.

Results

Patient characteristics

As described in Table I, fifteen patients participated in the study, with a median age of 71 years (range 56 – 87). The majority of the patients were males (80%). All enrolled patients had lower risk MDS, with 67% of the patients classified as int-1 and 33% as low-risk according to IPSS. Median marrow blast percentage on enrollment was 2% (range 0 – 5%). The majority of patients (11 out of 15, 73%) had diploid cytogenetics; 2 had del 20q; 1 had Y-; and 1 had del 5q. All patients were transfusion dependent at enrollment: 6 (40%) required red blood cells (PRBC) and platelets, 8 (53%) required only PRBC, and 1 (7%) required only platelets. All patients had at least one prior therapy with the median number being 2 (range 1–4), and 12 out of 15 patients (80%) had failed a hypomethylating agent (HMA). Ten patients were evaluated with NGS and 5 patients had qPCR for selected genes (CEBPA, FLT3, NPM1, IDH1, IDH2, JAK2, RAS, TP53, and c-KIT). The most frequently mutated genes were TET2 and JAK2V617F and occurred each in 2 patients. ASXL1 and TP53 were mutated in one patient each and only one patient (Patient 13) harbored 2 mutations: TP53 and TET2.

Table I.

Patients characteristics.

Clinical parameter (n=15)	Value (%)
Median age [range]	71 yrs. [56–87]
Sex
Male	12 (80)
Female	3 (20)
IPSSa category
Low risk	5 (33)
Intermediate-1	10 (67)
Median blast percentage [range]	2% [0–5]
Cytogenetics
Diploid	11 (73)
Complex	0 (0)
Other (2 del 20q, 1 Y-, 1del 5q)	4 (27)
Median number of prior therapies [range]	2 [1–4]
Prior HMAb failure	12 (80)
Transfusion dependence
RBCc	8 (53)
Platelets	1 (7)
Both	6 (40)

^aInternational prognostic system.

^bHypomethylating agents

^cRed blood cells.

Clinical response

At the median follow up of 22 weeks, the overall response rate was 20% with 3 patients achieving HI-E with a mean duration of response of 14 weeks (range 4–21). The median number of cycles administered was 8 (range 1–14). Eight patients (53%) had stable disease, and 4 (27%) progression. One patient achieved red cell transfusion independence, and went from an average of four units of packed red blood cells transfusion dependence required every 2 weeks, to transfusion independence after 2 cycles. This transfusion independence lasted 18 weeks. The 3 responders were previously treated with a hypomethylating agent. Eventually, all patients were removed from the study, mostly due to an increase in transfusion requirements (7 out of 15 patients). Two patients were removed for worsening cytopenias, one patient for lack of response, and another patient due to an increase in marrow blasts from 3 to 12%. Two patients requested removal from the study; one of them for travel-related issues. One patient was removed due to a grade 2 neuropathy and one patient died from congestive heart failure exacerbation, which was unrelated to the study. With a median follow up of 31 months, median EFS was 3.9 months, median OS was not reached, and at 3 years 55% of the patients were alive (Supplementary Figure 1A and 1B). No correlation between response to treatment and cytogenetic and molecular genomic studies was identified.

Toxicities

Most adverse events were grade 1 or grade 2. The different types of adverse events and their respective grade levels are detailed in Supplementary Table I. Three patients experienced a local reaction at the subcutaneous injection site (redness or bruising), all grade 1. Seven patients experienced neuropathy, four grade 1 and three grade 2. The latter three patients required a dose reduction of bortezomib to 1.0 mg/m2 for persistent neuropathy. Three patients had grade 2 elevations in bilirubin levels. Three patients had grade 2 infections (febrile neutropenia, urinary tract infection (UTI), pneumonia). Nausea, vomiting, diarrhea, fatigue and rash also occurred in a few patients and were rated grade 1 or 2. Four patients experienced at least one grade 3 toxicity, which was attributable to bortezomib (renal dysfunction, nausea/vomiting, diarrhea and fatigue). One patient had congestive heart failure exacerbation and another patient experienced a cerebrovascular accident; however, these adverse events were not attributable to bortezomib.

Correlative studies

The median pp65 level at baseline was 31% (range 7 – 70%), which decreased to 23% by the end of cycle 1. The pp65 level decreased by at least 10% in 7 out of 13 evaluable patients (54%, p=0.025) (Supplementary Table II). This downregulation was at least a 50% decrease from baseline in 4 out of 7 cases. In six out of 7 patients, the decrease was noted by the end of cycle 1. In the remaining patient, the decrease occurred by the end of cycle 3 (Supplementary Table II). Interestingly, 3 responders were among the 7 patients who had a decrease in pp65 level. The responders had at least a 50% reduction in pp65 level from baseline, which was noted at the end of cycle 1 in two of the patients (pp65 40% to 10% and 40% to 20%), and at the end of cycle 3 in one patient (pp65 25% to 5%) (Figure 1A and Supplementary Figure 2). Clinical responses lasted between 4–21 weeks with a mean of 14.3 weeks. Eventual loss of response in these patients was also accompanied by an increase in pp65 level (Figure 1A). In the non-responders the pp65 level was variable with no particular pattern of change: it increased in seven patients, decreased in two patients (55% to 23% and 70% to 5%) and remained unchanged in one patient (15%) (Figure 1B and Supplementary Figure 2). The remaining two non-responders had a suboptimal sample and immunofluorescence could not be performed post-treatment.

Figure 1.

Changes in Pp65 levels over course of therapy.

A) Pp65 levels in Responders over the course of therapy.

B) Pp65 levels in Non-Responders over the course of therapy.

Cytomorphological analysis

Bone marrow characteristics are summarized in Table II. Bone marrow aspirates were reviewed to evaluate cellularity, blast count, iron immunohistochemistry stain for ring sideroblasts (RS), dysplasia (defined by MDS dysplasia score as described by Della porta et al.[54]), and nuclear factor kappa B (NF-κB) activity as measured by phosphorylated P65 (pp65) immunofluorescence. These characteristics were compared between the bone marrow (BM) sample at the beginning of the clinical trial and the last BM during treatment. One patient (Patient 4) had a suboptimal BM at the last assessment.

Table II.

Bone marrow characteristics.

		WHO Classificationa	Cellularity (%)		Blast (%)		Ring Sideroblast (%)		Della Porta MDS Dysplasia Score43		Pp65 (%)b			Cytogenetics		Somatic mutations
	Patient	-	Baseline	Post- Treatment	Baseline	Post- Treatment	Baseline	Post- Treatment	Baseline	Post- Treatment	During Treatment			Baseline	Post-Treatment	Baseline
											1st	2nd	3rd
RESPONDERS	1	RAEB-1c	100	90	3	3	60	47	9	5	25	5	56	45,XY,[20]	45,XY,[20]	None
	7	RCMDd	50	100	1	2	0	0	6	5	40	10	22	45,XY,[20]	45,XY,[20]	JAK2: 3%
	12	RAEB-1	80	65	2	1	43	30	8	3	40	20	22	45,X,-Y[20]	45,X,-Y[20]	ASXL1
NON-RESPONDERS	2	CMML-1e	90	70	3	12	0	0	3	9	15	NAf	15	45,XY,[20]	46,XY,del(13)(q12q14)[3]46,XY[17]	NA
	3	RARSg	95	95	2	1	40	40	9	4	28	50	50	45,XY,[20]	45,XY,[20]	NA
	4	RCMD	NA	NA	5	4	3	NA	9	10	43	NA	NA	45,XY,[20]	NA	None
	5	RARS	60	60	1	0	95	83	9	9	7	NA	35	45,XY,[20]	45,XY,[20]	None
	6	RCMD	90	100	1	3	85	45	6	3	25	35	48	45,XY,[20]	NA	TET2
	8	RCMD	25	80	1	1	6	NA	5	9	20	18	41	46,XY,del(20)(q11.2q13.3)46,XY,[10]46,XY[10]	46,XY,del(20)(q11.2q13.3)46,XY,[18]46,XY[2]	None
	9	RCMD	100	100	0	2	NA	NA	9	6	15	11	15	45,XY,[20]	45,XY,[20]	None
	10	RCMD	90	90	2	0	3	NA	7	2	55	NA	23	47,XY,del(20)(q11.2q13.3), +21[20]	47,XY,del(20)(q11.2q13)+21[20]	RUNX1
	11	RCMD	60	60	3	2	18	1	6	6	70	0	5	45,XY,[20]	NA	NA
	13	MDS isolated del(5q)h	10	65	3	6	NA	NA	8	9	10	20	26	46,XY,del(5)(q15q33)[20]	46,XY,del(5)(q13q33)[5]/46,idem,del(12)(p12p13)[4]/46,idem,der(12)t(12;22)(p11.2;q11.2), der(22)t(12;22)del(12)(p11.2)[9]/46,idem,t(4;22)(q31;q11.2)[2]	JAK2:30% TP53 <5%
	14	CMML-1	NA	NA	1	4	6	0	6	7	50	NA	NA	45,XY,[20]	NA	NA
	15	RCMD	70	80	2	3	60	51	6	8	20	NA	25	45,XY,[20]	NA	TET2

^aWHO: World health organization 2008

^bpp65: phosphorylated-p65 (pp65) levels

^cRAEB-1: Refractory anemia with excess of blast-1

^dRCMD: Refractory cytopenia with multilineage dysplasia

^eCMML-1: Chronic myelomonocytic leukemia type 1

^fNA: Not available

^gRARS: refractory anemia with ring sideroblast

^hMDS isolated del(5q): myelodysplastic syndrome associated with isolated del(5q)

The responder group included 2 patients with previously treated RAEB-1 (Patient 1: 5% blast and Patient 12: 6% blast at initial diagnosis) and one RCMD (Table II). In the responder group (n=3), all patients showed improvement in bone marrow dysplastic findings. Patient 1 and 12 also had a decrease in ring sideroblasts (RS) count (60 to 47% and 43% to 30%, respectively). Of interest, patient 7 had an increase in cellularity, (50% to 100%) and marrow fibrosis (MF-2 to MF-3). The morphologic changes correlated with the reduction in pp65 level in the second sample during treatment, and Patient 1 showed an increase in pp65 level (from 5% to 56%) at the time of treatment failure. No changes in bone marrow blast count and cytogenetic studies were noted (Table II and Supplementary Figure 3).

In the non-responder group (n=11), four patients (Patients 3, 6, 9 and 10) showed an improvement in dysplasia. One patient (Patient 10) also had an associated decrease in pp65 level from 55% to 23%. Other patients had an increase in pp65 level or a stable level (Table II). Patient 8 showed worsening dysplasia with an associated increase in pp65 level (from 20% to 41%). Patient 4 also had worsening dysplasia, but pp65 level could not be done at the last BM assessment due to suboptimal sample. Patients 14, 2 and 13 had increase in blasts (1% to 4%; 3% to 12% and 3% to 6%, respectively). None of them showed a decrease in pp65 level. RS was decreased in 5 patients (Patients 5, 6, 11, 14 and 15). This reduction in RS count correlated with pp65 decrease only in Patient 11. The remainder of patients had no change in pp65 level.

From the non-responder group (n=11), seven patients had available cytogenetics in both samples (Patients 2, 3, 5, 8, 9, 10 and 13). From that group, 5 patients had no change in karyotype and 2 patients showed new cytogenetic aberrancies. One patient (Patient 2) acquired a new clone with del(13q) and the other (Patient 13) had clonal evolution from del(5q) to complex karyotype. These findings correlated with blast increase in both cases. Patient 13, who evolved to complex karyotype, also had an increase in pp65 level from 10% to 26% and was the only patient who harbored 2 mutations that included TP53 at the first assessment.

Effect on Ring sideroblasts

Unexpectedly, we observed a decrease in the number of ring sideroblasts (RS). While comparing bone marrow samples at baseline and after treatment, a trend emerged regarding the number of ring sideroblasts. The majority of evaluable patients had a decrease in RS while on study. Five patients were not evaluable. Out of 10 evaluable patients who had RS assessment available at both time points, 7 patients (70%) had a significant decrease in RS count (p=0.017). This effect was observed regardless of clinical response. Among the responders, 2 out of the 3 patients had a decrease in RS (60 to 47% and 43% to 30%). Among the non-responders, 5 out of 7 evaluable patients had a decrease in RS (95 to 83%, 85 to 45%, 18 to 1%, 6 to 0% and 60 to 51%). This change was seen not only in RARS. In fact only 2 patients were classified as having RARS, both of whom were in the non-responder group and only one of them had a decrease in RS count (95 to 83%) (Table II and Figure 2).

Figure 2.

Bone marrow ring sideroblast (RS) changes with bortezomib treatment. Left panels represent BM at baseline. Right panels represent the last BM during treatment. Iron stain (magnification, 1000×).

A) Decrease in ring sideroblast counts from 95% to 83% in patient 5.

B) Decrease in ring sideroblast counts from 85% to 45% in patient 6.

C) Decrease in ring sideroblast counts from 60% to 51% in patient 15.

Discussion

Compelling data now support a deregulation of the innate immune system and inflammatory signaling in the pathogenesis of MDS.[5–11] The evidence implicates activation of TLR signaling, culminating in upregulation of the NF-κB pathway, as an important molecular mechanism in MDS.[8–11] Bortezomib is a proteosomal inhibitor known to induce apoptosis of malignant hematopoietic cells, at least partly by downregulating NF-κB. [23–25] We designed this proof of concept study and tested bortezomib in low and int-1 risk MDS patients. This study differs from other studies using bortezomib in MDS in that it selectively targets patients who had evidence of NF-κB activation in their bone marrow by using pp65 quantification as a surrogate marker.

From a clinical standpoint, our study showed that SC bortezomib was overall well-tolerated and safe to use in patients with lower-risk MDS. It also showed that SC bortezomib had modest clinical activity in this patient group and resulted in hematologic improvement in 20% of patients with lower risk MDS who had evidence of NF-κB activation in their bone marrow. Notably, the patients who benefited most had previously failed hypomethylating agents (HMA). Previous studies demonstrated that bortezomib has hypomethylating activity via down-regulation of DNA methyltransferase (DNMT) expression, rather than by direct enzymatic inhibition,[56] which could partly explain the activity of bortezomib even after resistance to HMA. There was no correlation between clinical response and MDS-related molecular or cytogenetic abnormalities; however, the small number of patients limits the analysis.

From a pathologic standpoint, the findings on a cellular and molecular level were very interesting. All patients with response to bortezomib therapy had a decrease in the MDS dysplasia score. This improvement in dysplastic features was seen in megakaryocyte, granulocyte, and erythrocyte cell lines. One of the most intriguing findings is the significant decrease in ring sideroblasts noted in 70% of evaluable patients. This finding could be explained by the fact that the proteasome plays an important role in iron homeostasis [57, 58] that may be altered by proteasomal inhibition using bortezomib. The proteasome mediates the degradation of Iron Regulatory Proteins (IRP2 and in some cases IRP1), which play an essential role in maintaining intracellular iron balance by regulating the translation of ferritin and transferrin receptors.[59] Therefore it is reasonable to think that bortezomib could have an effect on the mitochondrial ferritin accumulation as evidenced by reduction in ring sideroblasts. This is a novel finding that has not been previously reported, and if confirmed in a larger patient population could have implications on the treatment of patients with RARS who harbor SF3B1 mutations using proteasome inhibitors.

The other interesting pathologic finding is the modulating effect of bortezomib on NF-κB activity. Bortezomib caused a decrease in the level of phosphorylated p65, a surrogate for NF-κB activation, in 7 out of 13 evaluable patients (54%, p=0.025). Interestingly, significant downregulation correlated with clinical response; however, the clinical responses were short-lived and eventual loss of response correlated with increase in pp65 level. Hence, one could argue that a drug with a more potent and a more specific inhibition of the NF-κB pathway could lead to a more significant and long-lasting downregulation of pp65 and hence, could lead to a better clinical response rate. Bortezomib induces apoptosis of malignant hematopoietic cells partly through NF-κB inhibition, but it also affects many other pathways through proteosomal inhibition and therefore, is not a specific inhibitor of the NF-κB pathway. Specific inhibitors of this pathway, notably inhibitors of IκB kinases (IKKs), are being developed with some undergoing preclinical evaluation and others already being used in clinical trials.[60–63] In the future, these selective inhibitors of NF-κB could be tested for use in MDS.

In conclusion, our study provides new insights regarding what happens on a cellular and molecular level when using SC Bortezomib in lower risk MDS patients. We suggest that proteasomal inhibition could have a role in the treatment of RARS in the future. We also suggest that NF-κB activation, measured by pp65 level, could be a useful biomarker in select patients with lower-risk MDS who could benefit from therapies that target this pathway. Further studies are needed to validate these findings and to ascertain pp65 as a predictive biomarker.

Supplementary Material

Supp Table&Figure

Supplementary Table I: Toxicity assessment.

Supplementary Table II: Quantification of pp65 levels.

Supplementary Figure 1: Survival curves

A) Event free survival.

B) Overall survival.

Supplementary Figure 2: Immunofluorescence staining (pp65).

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  Top panel: positive and negative controls.
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  Middle panel: MDS patient with progression of disease and increase in pp65 levels from 28% at baseline to 50% at last BM assessment.
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  Bottom panel: patient with initial response followed by progression with increase in pp65 levels: baseline pp65 level: 25%, during treatment pp65 level 5% (initial response), last pp65 level: 56% (progression).

Supplementary Figure 3: Bone marrow morphologic changes with bortezomib treatment. (Patient 1). Left panels represent BM at baseline. Right panels represent the last BM during treatment.

A) Assessment of megakaryocytic dysplasia; from hypolobated dysplastic megakaryocytes at baseline to megakaryocytes with multilobated nuclei in the last follow up. (Hematoxylin and eosin stain; magnification 400×).

B) Assessment of granulocytic and erythroid dysplasia. At baseline hypogranular and pseudo pelger-hüet granulocytes and erythroid cells with nuclei budding. In the last follow up: granulocytes with proper granulation and adequate nuclei lobulation and erythrocytes with cytoplasm with uneven hemoglobinization. (Wright-Giemsa stain; magnification 400×).

C) Iron stain with 60% ring sideroblast at baseline decreased to 47% at last assessment (magnification, 1000×).