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Allelic deletion of the RPS14 gene is a key effector of the hypoplastic anemia in patients with myelodysplastic syndrome (MDS) and chromosome 5q deletion (del(5q)). Disruption of ribosome integrity liberates free ribosomal proteins to bind to and trigger degradation of mouse double minute 2 protein (MDM2), with consequent p53 transactivation. Herein we show that p53 is overexpressed in erythroid precursors of primary bone marrow del(5q) MDS specimens accompanied by reduced cellular MDM2. More importantly, we show that lenalidomide (Len) acts to stabilize MDM2, thereby accelerating p53 degradation. Biochemical and molecular analyses showed that Len inhibits the haplodeficient protein phosphatase 2A catalytic domain alpha (PP2Acα) phosphatase resulting in hyperphosphorylation of inhibitory serine-166 and serine-186 residues on MDM2, and displaces binding of RPS14 to suppress MDM2 autoubiquitination whereas PP2Acα overexpression promotes drug resistance. Bone marrow specimens from del(5q) MDS patients resistant to Len overexpressed PP2Acα accompanied by restored accumulation of p53 in erythroid precursors. Our findings indicate that Len restores MDM2 functionality in the 5q- syndrome to overcome p53 activation in response to nucleolar stress, and therefore may warrant investigation in other disorders of ribosomal biogenesis.

CONTEXT AND OBJECTIVE: Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) is a provisional entity included in the MDS/MPN overlap syndromes, with features of refractory anemia with ring sideroblasts (RARS) and thrombocytosis (platelet count ³ 450 x 10/L), recently renamed MDS/MPN with ring sideroblasts and thrombocytosis. Management of RARS-T is extrapolated from MDS and MPN treatment options namely for alleviating symptoms of anemia and, occasionally, for thrombocytosis. Individual case reports have suggested that lenalidomide may be effective in treating RARS-T. In non-del 5q, low-risk MDS, 25% of patients (pts) became red blood cell transfusion independent (RBC-TI). In the phase II study of lenalidomide in non-del 5q lower risk MDS, 35% of pts with RARS treated with lenalidomide achieved RBC-TI. The primary objective of this study is to evaluate a series of pts with RARS-T and compare hematological improvement (HI) responses among different treatment options. METHODS: This is a retrospective, single-center study. Patients with RARS-T were identified from the MDS Database at Moffitt Cancer Center. Samples for somatic mutation testing were obtained at time of referral. Descriptive statistics were used to describe baseline characteristics and response rates to treatment. The primary endpoint was HI using international working group criteria (IWG) 2006. The median overall survival (OS) was calculated from time of diagnosis. RESULTS: We identified 33 pts with RARS-T. The mean age was 67 years (38-80), 15 pts were male (45.5%), and majority were Caucasians (n=27, 82%). According to international prognostic scoring system (IPSS), 24 patients (73%) were low risk and 9 pts (27%) were int-1 risk. By revised IPSS, 11 pts (33%), 18 (55%), 3 (9%), and 1 (3%) were very low, low, intermediate, and high risk, respectively. The mean WBC was 7.7 (2.8-25.7), the mean Hemoglobin was 9.9 g/dl (7.3-12.4), and the mean platelet count was 682 (372-1644). Among 20 pts who had Next generation sequencing for somatic mutations at time of referral, 19 pts (95%) had SF3B1 mutation and 4 pts (20%) had JAK-2 V617 F mutation. The median OS was 90 mo (95% CI 47-134). For pts with JAK-2 V617F mutations, the median OS was 110 mo versus 74 mo for those with wild type (p=0.24). The rate of AML transformation was 3% (one patient only). Among the 33 pts, 8 pts had no treatment (observation only), 22 pts (67%) received erythroid stimulating agent (ESA) prior to any active therapy, 2 patients received lenalidomide with no prior ESA, and 1 pt ruxolitinib with no prior ESA. Twelve pts (36%) received lenalidomide (7 as first line active therapy after ESA and 2 without prior ESA exposure) and 13 pts (33%) received azacitidine (6 as first line active therapy after ESA). The HI rate for ESA was 36% (12/22) with a median duration of ESA treatment of 33mo. The HI rate for lenalidomide was 50% (6/12) with a median duration of lenalidomide treatment of 10.3mo. Among the 9 pts who received lenalidomide as first line active therapy (2 pts without prior ESA exposure) HI rate was 56%. The HI rate for azacitidine was 15% (2/13) with a median duration of azacitidine treatment of 6.3mo. CONCLUSIONS: Pts with RARS-T had favorable outcomes, with long overall survival and a low rate of AML transformation. Rates of hematological improvement with the use of azacitidine were lower than expected. Lenalidomide is an effective treatment option and should be considered when treating patients with RARS-T. Figure. Figure. Disclosures Sallman: Celgene: Research Funding, Speakers Bureau. List:Celgene: Research Funding. Komrokji:Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau.

Introduction: Glutathione S-transferase (GST) P1-1 binds to and inhibits Jun kinase (JNK), a key regulator of cellular proliferation, differentiation and apoptosis. TLK199, a glutathione analog, binds selectively to GSTP1-1 fostering dissociation from JNK, kinase activation and the promotion of growth and maturation of hematopoietic progenitors in preclinical models, while promoting apoptosis in human leukemia cell lines. The intravenous study with liposomal TLK199 resulted in hematologic improvement (HI) in MDS patients (pts); this trial utilizes an oral formulation of TLK199 in MDS pts. Methods: The objectives of this study were to determine the safety and pharmacokinetics (PK) of TLK199 Tablets given b.i.d. at total daily doses ranging from 200 mg to 6000 mg for the first 7 days of each 3-week cycle. The design was a standard 3 by 3 (3 pts per dose level) dose escalation. Patients were treated until MDS progression or unacceptable toxicity up to a maximum of 8 cycles. Six pts underwent fed-fast PK analysis to determine the effect of food absorption. The PK was evaluated over the dose range for TLK199, and metabolites TLK236, TLK235, and TLK117. Results: 44 MDS pts (32 M/12 F), (9 RA, 11 RARS, 3 RAEB/RAEB-1, 1 RAEB-II, 7 RCMD, 2 RCMD-Rs, 2 CMML, 5 MDS-U, 4 Unknown); IPSS risk-low 14 (32%), INT-1 27 (61%), and INT-2 3 (7%); median age 72 years (range 53–84), received total 206 cycles, median 4.5 (range 1–9) cycles/pt. Ten pts (23%) completed the intended 8 cycles of therapy. Two pts had dose reductions and 4 pts had dose delays (2 due to adverse event (AE) and 2 for scheduling difficulty) at single cycle. Twenty-seven pts (61%) were red cell transfusion (tx) dependent and 5 pts (11%) were platelet tx dependent. Sixteen pts (36%) had abnormal karyotypes. Most common treatment-related AEs were non-hematologic: There were no Grade 3 or 4 toxicities; Grade 2 toxicities were diarrhea and nausea in 2 pts each (5%). Grade 1 toxicities were nausea (43%), diarrhea (25%), vomiting (18%), abdominal pain (7%) and constipation (7%). Three pts (7%) experienced pill-induced dysphagia and reflux esophagitis. Two pts (5%) had Grade 4 neutropenia and 1 pt had febrile neutropenia. There were no DLTs reported. The plasma concentration of the primary active metabolite, TLK236, increases with TLK199 Tablets dose with a mean t1/2 = 2.5 h (range 1.5–4); Cmax = 4.5 uM (range 0.4–6.3). There was no difference seen in the fed vs. fasted patients. By IWG criteria, 15 individual cell line HI responses were observed at the various dose levels of 1000–6000 mg/day with 9 HI responses at dose levels 4000–6000 mg/day. These HI responses were characterized as 1 HI-E major and 4 HI-E minor, 1 HI-N major and 2 HI - N minor, 1 HI-P major and 6 HI-P minor. One bilineage response was reported at 5000 mg/day and 2 trilineage responses at 6000 mg/day. These responses were accompanied by clinical symptoms improvement. Conclusions: TLK199 Tablets are well tolerated and HI responses in all three cell lines were observed with oral TLK199 short-course schedule. These data support the Phase 2 development of extended schedules of oral TLK199 in MDS.

Reduced intensity conditioning has decreased mortality in HCT and has increased the age of eligible patients (pts), but has increased risk of post-transplant relapse. One strategy to decrease the risk of relapse in MDS pts with IPSS > Int-1 who benefit from immediate HCT is pre-transplant treatment with 5-azacitidine. We analyzed outcomes of 22 pts who received a median of 3 (1–7) cycles of 5-azacitidine (AZA) and 13 pts who received supportive care alone (NO-AZA) prior to allogeneic HCT. Pts who received other chemotherapy for treatment of MDS were excluded. Pts received targeted busulfan (3500–6000 AUC) and fludarabine conditioning followed by peripheral blood HCT from an HLA sibling or unrelated donors between July 2004 and April 2007. Pts received GVHD prophylaxis consisting of tacrolimus plus either methotrexate or mycophenolate mofetil. AZA pts were significantly older [median age 56 (range 40–69) yr] than the NO-AZA pts [median age 49 (range 34–63) yr] (p=0.03). At referral to HCT and before starting 5-azacytidine, 20 AZA pts had MDS at Int-1 (8), Int-2 (9), or High (3) IPSS risk category; and 1 pt each had CMML-1 and CMML-2. The 12 NO-AZA pts had MDS Low (1), Int-1 (3), and Int-2 (5); 2 pts did not have karyotype information available, and one had CMML-1. Prior to HCT, AZA pts had MDS Low (4), Int-1 (6), Int-2 (7) or High (3), and 1 pt each had CMML-1 and CMML-2; NO-AZA pts had MDS Low (1), Int-1 (4), Int-2 (5) and High (1), and one had CMML-2 (p=0.8). Cytogenetics were not available in 1 pt. Donors for AZA pts were HLA-identical siblings (6), HLA-A, B, C, DRB1 matched unrelated (13), or single locus mismatched unrelated (3). Donors for NO-AZA pts, were HLA-identical siblings (8), matched unrelated (4), or a single locus mismatched unrelated (1) (p=0.06). Post-transplant follow-up for surviving pts is a median of 13.6 (range 3.8–31.2) vs 25.6 (range 3.8–34.7) months, respectively for AZA vs. NO-AZA pts (p=0.12). All evaluable AZA pts achieved complete remission posttransplant. In the NO-AZA pts, 1 pt had persistent MDS at the time of his death on day 62; the remainder achieved complete remission. At 100 days, the cumulative incidence of grade 2–4 GVHD was 92% vs 68% and grade 3–4 GVHD 36% vs. 8% in AZA vs NO-AZA, respectively, but the apparent differences can be at least in part explained by the higher prevalence of unrelated donors used for AZA pts and their higher age. Using a Cox proportional hazards regression model including age and donor, the use of 5-azacitidine was not a significant predictor for GVHD. At 2 years after HCT, the cumulative incidence of relapse was 45% vs 37%, in AZA vs NO-AZA (p=ns), and overall survival was 43% vs. 59%, in AZA vs NO-AZA (p=ns). Causes of non-relapse death in AZA include relapse of pre-existing NHL (1), GVHD (2), infection (1 viral, 1 fungal), interstitial pneumonitis (1), sudden cardiac death (1), and unknown (1) and in NO-AZA pts include GVHD (1), and multiorgan failure (1). From these observations it appears that pretransplant treatment with 5-azacitidine may not be associated with a lower risk of relapse posttransplant. However, 5-azacitidine may be of value in stabilizing disease thereby allowing time for pts who may otherwise experience progressive disease to reach transplant. Further appropriately designed studies will be needed to address the safety of this strategy.

Most patients with MDS are older than 60 yrs and, because of age, many have previously been excluded from HCT due to the high risk of transplant related mortality. Reduced intensity conditioning has decreased regimen related mortality in HCT and has increased the age of patients for whom this treatment is offered. There remains substantial risk of relapse after HCT especially in patients with > Int-1 MDS who benefit the most from immediate HCT. One strategy to suppress leukemia burden and potentially decrease the risk of relapse is pre-transplant treatment with 5-azacitidine (Vidaza®). This DNA hypomethylating drug is FDA approved for the treatment of MDS. We analyzed the outcome for 34 MDS patients who received a myeloablative HCT from an HLA compatible sibling or unrelated donor from July 2004 through June of 2006, to investigate the potential impact of pre-transplant 5-azacitidine on response and post-transplant relapse. Fourteen of thirty-four patients received a median of 2 (1–7) cycles of 5-azacitidine prior to HCT. Median age was 59 (49 – 69) yrs. Diagnosis and IPSS category included MDS (8) [Int-1 (1) and Int-2 (7)], MDS-AML (5) and CMML (1). Two patients had induction chemotherapy with residual MDS prior to 5-azacitidine. Subsequent to 5-azacitidine: ten responded [CR (2), PR (1) improvement (7)] three progressed and one was not evaluable. The IPSS category after 5-azacitidine treatment and prior to HCT was Int-2(1), Int-1 (4), and Low (5; 3 in CR) in addition to AML (2) and CMML (1). One was unable to assess. The median follow-up in the survivors is 191 (47 – 524) days. Of the fourteen 5-azacitidine treated patients, none have relapsed post-HCT including patients who progressed on 5-azacitidine. Four have died from non-Hodgkins lymphoma (1), DAH (1), adenovirus pneumonia (1) and chronic GVHD (1). Ten are alive in remission, two for greater than one year. Twenty patients did not receive 5-azacitidine. Diagnosis and IPSS category included MDS (9) [Int-1 (3) and Int-2 (6)], MDS-AML (9) and CMML (1) and one not determined due to lack of cytogenetics. Their median age was 53 (33 – 68) yrs. Treatment included supportive care, induction chemotherapy (9) and SCIO-469 (1). IPSS distribution prior to HCT was Int-2 (7), Int-1 (5) Low (5; 4 in CR); in addition to AML (1) and CMML (1). One was unable to assess. The median follow-up in the survivors is 420 (21 – 742) days. Ten of twenty are alive without relapse and eight have relapsed. Five have died, with relapse (3), fungal pneumonia (1) or multi-organ failure (1). The 1 year K-M estimates of overall and progression free survivals are 64% (SEM 15%) and 64% (SEM 15%), respectively for 5-azacitidine group and 70% (SEM 11%) and 51% (SEM 13%) for non-5-azacytidine group. We conclude from this preliminary analysis that pre-HCT conditioning treatment with 5-azacitidine may reduce the risk for MDS relapse after allogeneic transplant in higher risk patients. In addition to its direct anti-tumor effect, 5-azacitidine may sensitize neoplastic cells to the effects of high dose chemotherapy or promote the expression of antigens critical to effective graft-vs-tumor response. This treatment strategy will be evaluated in a prospective trial to investigate the role of pre-transplant 5-azacitidine on transplant outcomes in patients with higher risk MDS.

Disease-free survival (DFS) is short in patients ≥ age 60 or with secondary AML, adverse cytogenetics, or leukostasis at presentation. In such patients, median CR duration is ≤ 8 mos and only 20% achieve 1 yr DFS. Historically, maintenance therapy with low doses of cytotoxic chemotherapy has not prolonged DFS. We tested the hypothesis that Tipifarnib (T) might be active as maintenance therapy for adults with poor-risk AML in first CR after induction and consolidation therapies. Oral T 400 mg bid for 2/3 wks was begun median 2.4 mos (range 1.0–4.1) after start of final consolidation cycle and given for up to 48 wks (16 cycles) to 36 adults with median age 63 (range 27–82), secondary AML 31%, adverse cytogenetics 47%, leukostasis 17%, ≥ 2 risk factors 40%. T was well-tolerated, with only 4 of 36 unable to complete 2 cycles because of constitutional symptoms (1 rash, 3 non-compliant). To date, 256 cycles have been administered (median 8 per pt, range 1–16), with hospitalization required during only 4 (1.5%) cycles (infection 3, bowel obstruction 1). Dose reductions for myelosuppression (400 mg bid to 300 mg bid) occurred in 17/32 (53%) by cycle 3, and 2 (6%) needed platelet transfusions. A total of 9 patients completed all planned 16 cycles of T with a median CR duration of 24 mos (range 15–36+). Five of the 9 remain in continuous CR (CCR) 19+-36+ mos, median 26+), compared with 4 who relapsed after CR of 15–24 mos (median 22). There are 8 additional pts in CCR and still receiving T (2–12 cycles) with CCR 4+-12+ mos. A total of 15 patients progressed while on T at median 6.5 mos CR (range 3.5–12). Median CR duration for all patients is 10+ mos (range 3.5–36+), with 88% ≥ 6 mos and 48% ≥ 12 mos. In 13 “comparable” poor-risk pts (age ≥ 60 46%, secondary AML 25%, adverse cytogenetics 46%, leukostasis 23%) who were eligible for but declined T, 4 are in unmaintained CCR at 14+-25+ mos, 9 have relapsed at median 7.5 mos (5–13 mos). Treatment with T did not have a negative impact on reinduction chemotherapy at relapse, as 6 of 9 patients achieved second CR. Administration of T in CR after induction and consolidation therapy has low toxicity and is associated with prolonged DFS in some adults with poor-risk AML. Phase 3 studies are warranted in this patient population.