Lasho, Terra L.

Abstract Introduction: Primary myelofibrosis (MF) is a chronic clonal malignancy which is compounded by the presence of inflammation, bone marrow fibrosis/scarring and risk of transformation to acute myeloid leukemia (AML). Ruxolitinib (Rux), a JAK2 inhibitor, is beneficial for reduction of symptoms and splenomegaly, however, significant myelosuppression is also a toxic side effect of this treatment, limiting optimal drug dosage in many patients. 9-ING-41 is a small molecule GSK-3β inhibitor which has significant clinical activity in patients with advanced malignancies and causes no myelosuppression. We thus assessed the effect of 9-ING-41in MF alone or in combination with Rux. We performed ex-vivo colony assays with primary cells from cases with untreated MF along with normal bone marrow (BM). The experiments were designed to assess the effect of drug on the ability of MF stem/progenitor cells to proliferate/differentiate by evaluating the number, size, and the morphology of colonies, both with and without treatment. Methods: Primary peripheral blood mononuclear cells from MF patients and BM from controls were plated in methylcellulose in duplicate, containing cytokines promoting hematopoietic growth, in the presence of either no drug, Rux only (0.05µM), 9-ING-41 only (range: 0.125µM - 10µM), or a combination of 9-ING-41 (range: 0.125µM - 10µM) and Rux (0.05uM). Colonies were counted ten days later, and colony growth frequency, distribution, and morphology were calculated. Results: We observed an increasing presence of large primitive granulocytic/erythroid/macrophage/monocyte (GEMM) colonies proportional to no drug treatment and linear to increasing concentrations of 9-ING-41 in both MF cases relative to other colony types and normal BM, suggestive of a selective primitive proliferative and/or differentiation effect of GSK-3β inhibition on MF hematopoietic progenitor growth. We did not observe this effect in the combinatory treatments (9-ING-41 + Rux), and there was no significant difference on MF colony number or size in colonies grown without treatment or with Rux alone (Figure 1 A/C/E). However, the morphology of the colonies within different treatments was remarkable. In both MF cases, either without treatment or addition of Rux only (0.05µM), the colonies appeared similar to each other -irregular and disorganized relative to normal bone marrow (Figure 1, B/D/F). In the presence of 9-ING-41 alone, we observed morphologically very large, fully differentiated primitive colonies in both MF cases. However, the most striking observation was the comparison of colonies after combination of both inhibitors. These colonies appeared normal and healthy-looking -discreet, round size and similar in morphology to normal bone marrow colonies. Additional studies are currently underway to look at the signaling consequences, including proliferation, differentiation, and cell cycle effect. Our preliminary results suggest this same mechanism might be occurring in MF cases and could explain the selective presence of large differentiated primitive colonies in MF vs. normal BM in the presence of 9-ING-41. Combining 9-ING-41 with Rux may reduce the dose needed for optimal therapeutic response and/or reverse myelosuppression. A Phase 2 study of 9-ING-41 in patients with advanced MF will be conducted. Citation Format: Terra L. Lasho, Francis Giles, Christy Finke, Naseema Gangat, Ayalew Tefferi. 9-ING-41, a GSK-3β-selective small molecule inhibitor, in combination with ruxolitinib in JAK2617F primary myelofibrosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5217.

Abstract Large-scale sequencing studies of hematologic malignancies have revealed notable epistasis among high-frequency mutations. One of the most striking examples of epistasis occurs for mutations in RNA splicing factors. These lesions are among the most common alterations in myeloid neoplasms and generally occur in a mutually exclusive manner, a finding attributed to their synthetic lethal interactions and/or convergent effects. Curiously, however, patients with multiple-concomitant splicing factor mutations have been observed, challenging our understanding of one of the most common examples of epistasis in hematologic malignancies. In this study, we performed bulk and single-cell analyses of patients with myeloid malignancy who were harboring ≥2 splicing factor mutations, to understand the frequency and basis for the coexistence of these mutations. Although mutations in splicing factors were strongly mutually exclusive across 4231 patients (q < .001), 0.85% harbored 2 concomitant bona fide splicing factor mutations, ∼50% of which were present in the same individual cells. However, the distribution of mutations in patients with double mutations deviated from that in those with single mutations, with selection against the most common alleles, SF3B1K700E and SRSF2P95H/L/R, and selection for less common alleles, such as SF3B1 non-K700E mutations, rare amino acid substitutions at SRSF2P95, and combined U2AF1S34/Q157 mutations. SF3B1 and SRSF2 alleles enriched in those with double-mutations had reduced effects on RNA splicing and/or binding compared with the most common alleles. Moreover, dual U2AF1 mutations occurred in cis with preservation of the wild-type allele. These data highlight allele-specific differences as critical in regulating the molecular effects of splicing factor mutations as well as their cooccurrences/exclusivities with one another.

8542 Background: Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence of leukemia-associated somatic mutations in clonally expanded hematopoietic stem cells with a VAF of ≥2%. CHIP has been associated with myeloid malignancies and increased all cause mortality largely from atherovascular disease. The prevalence and impact of CHIP in patients with myeloma (MM) is yet to be defined. Aims: Evaluate the prevalence and significance of CHIP in 101 lenalidomide exposed patients with multiple myeloma. Methods: 101 MM patients, the majority exposed to > 2 years of lenalidomide (Len), with stored mononuclear blood samples were identified for Next Generation Sequencing (NGS) using a panel encompassing 42 gene mutations associated with CHIP. Electronic medical records were reviewed and outcomes of secondary malignancy, macrocytosis, thrombosis and mortality were extracted. Results: Thirty of 101patients were found to have CHIP with the most frequent mutations: DNMT3A (12%), TET2 (5%), and TP53 (4%). One third of patients with CHIP had > 1 mutation (37%). At 68 months median follow up over a quarter of patients experienced thrombosis (31%) and 13% developed subsequent malignancy/premalignant condition including myelodysplastic syndrome (3%). There was no significant difference in age, gender, duration of Len prior to NGS testing, thrombosis complication, or survival in those with versus without a CHIP mutation. At last F/U 30% had died, 25% remained on therapy, 27% were on maintenance therapy and 11% were on observation alone. Conclusions: CHIP mutations are common in plasma cell neoplasms, with 30% of patients in this study retrospectively found to have CHIP mutations by NGS testing. CHIP mutations have been associated with cardiovascular events and malignancy development, however no associations were identified in this small study. Limitations to this study include small sample size, retrospective nature and duration of follow up. Further study of this very common finding in MM patients regarding timing of development and subsequent impact of these mutations could help risk stratify and inform therapy selection. [Table: see text]

Abstract Background: We recently reported on MPLW515L/K mutations in 1182 patients with myeloid disorders (Blood, First Edition July 25, 2006) and found low frequency occurrence in in agnogenic myeloid metaplasia (AMM) (~5%) and essential thrombocythemia (ET) (~1%), but not polycythemia vera (PV). Of the 20 MPLW515L/K-positive patients in that series, 6 (30%) also carried a low burden of the JAK2V617F mutation. Studies of mutation prevalence in individuals with clonal granulopoiesis as well as familial myeloproliferative disorders (MPD) studies suggest that these mutations (JAK2V617F) may be acquired as secondary genetic events. Aim: to gain additional insight regarding the aforementioned two mutations and MPD pathogenesis by determining (i) concordance between mutation expression and clonal hematopoiesis, (ii) the instructive role of each mutation in determining lineage fate, iii) differences in pattern and lineage restriction of these mutations between various MPD, and (iv) chronology of clonal emergence and clonal dominance for the 2 mutations. Methods: Sixteen MPD patients were studied (13 JAK2V617F+ and 3 MPLW515L/K+); 9 had PV (all JAK2V617F+), 2 ET (both JAK2V617F+), 1 CMML (JAK2V617F+), and 4 AMM (2 MPLW515K+, and one each with MPLW515L and JAK2V617F). CD34+ progenitor cells were plated in methylcellulose and resulting hematopoietic colonies were scored on day 10–12. Colonies were obtained with (all patients) and without (10 patients; 5 with PV, 1 ET, and 4 AMM) cytokine support. Individual colonies were screened for presence of either mutation by DNA sequencing. In addition, MPLW515L/K analysis was performed in T and B lymphocytes in 3 informative cases. Finally, serially stored bone marrow was examined in 3 patients with co-expression of MPL515 and JAK2V617F mutations. Results: A total of 552 hematopoietic colonies from 16 MPD patients were genotyped. We found cohabitation of both mutation-positive and mutation-negative endogenous colonies in PV as well as other MPD. For JAK2V617F+ patients, the majority (75–100%) of erythropoietin-independent erythroid colonies (EEC) but not endogenous granulocyte colonies (20–70%) were mutation-positive. In contrast, no EEC were obtained from progenitors from the 3 MPLW515L/K+ patients. For 2 of the 3 patients, the endogenous myeloid colonies were predominantly mutation-positive (~90%). A mixed allele pattern (homozygous and/or heterozygous) was seen with both mutations regardless of MPD phenotype, but the overall mutant colony burden was higher with MPLW515L/K mutations relative to JAK2V617F. When such testing was possible, MPL515 mutations were detected in T-lymphocytes (2 of 3 patients) as well as B-lymphocytes (2 of 2 patients). Finally, among 3 patients carrying both mutations, serial marrow studies (25 time points over 4–8 years) revealed the occurrence of both mutations at diagnosis and persistent dominance of MPLW515L/K over JAK2V617F over time. Conclusions: Our observations provide direct evidence for clonal hematopoiesis that antedates acquisition of either MPLW515L/K or JAK2V617F that bias towards myeloid and erythroid fates, respectively. Furthermore, both mutations appear to occur early in the disease course and lineage studies suggest a hierarchically earlier stem cell origin for MPLW515L/K as compared to JAK2V617F.

Abstract Background: Constitutive activation of the JAK-STAT signaling pathway through acquisition of the JAK2V617F mutation plays a key role in the pathogenesis of related myeloproliferative disorders (MPD) including polycythemia vera (PV), agnogenic myeloid metaplasia (AMM), and essential thrombocythemia (ET). Hypothesis: Genetic variation in JAK2 and/or the cytokine receptor genes for erythropoietin (EPOR), thrombopoietin (MPL), and granulocyte colony stimulating factor (GCSFR), influences the expression of a specific disease phenotype (PV, AMM, or ET). Methods: We studied 179 Caucasian MPD patients (PV=84, AMM=58, ET=37) for genetic variation in 4 candidate genes (i.e. JAK2, EPOR, MPL, GCSFR) through single nucleotide polymorphism (SNP) and haplotype analyses. A total of 32 LD (linkage disequilibrium) tag-SNPs with a minimum allele frequency of at least 5% were selected from the HapMap CEU database (JAK2=13, EPOR=4, MPL=5, GCSFR=10). Genotyping was performed using archived DNA from enriched neutrophils. Results: Seventy six (94%), 26 (45%), and 14 (38%) patients with PV, AMM, and ET, respectively, carried the JAK2V617F mutation. Significant differences in allele frequencies was observed at 6 SNP loci (rs10758669, rs3808850, rs7849191, rs7046736, rs10815148, and rs12342421, p-values < 0.0005), all within the JAK2 gene, in comparing our overall study population with the founder Caucasian population in the HapMap database. In the additive genotype-phenotype association analysis adjusted for gender and age at diagnosis, 3 SNP loci in JAK2 (rs7046736, rs10815148, and rs12342421) were found to be significantly, but reciprocally associated with PV (p-values < 0.00006, odds ratio=0.37, 2.82, and 2.39, respectively) and ET (p-values < 0.002, odds ratio=2.50, 0.36, and 0.41, respectively), but not AMM, in terms of the minor allele being ’protective’. These three SNPs were all in high LD, with the ’r2’ measures of LD between 0.59 and 0.71. Furthermore, 2 additional JAK2 SNP loci (rs10758669, p-value = 0.0024 and rs10974947, p-value = 0.0046) were significantly associated with PV (odds ratio=1.88 and 0.47, respectively), but not ET or AMM. Similarly, presence of the minor allele at a single SNP locus in EPOR (rs318699, p-value = 0.0012) was significantly associated with PV only (odds ratio=2.16). For the phenotype-genotype intragene haplotype analyses, the EPOR intragene haplotypes GAAA and GGAA were significantly associated with PV (simulated global p-value = 0.058, simulated individual p-values 0.0013 and 0.0068, haplotype frequency of 35% and 56%, respectively). In addition to EPOR, 6 intragene haplotypes within JAK2 where significantly associated with PV (simulated global p-value < 0.0001, individual simulated p-values < 0.03). Conclusion: The current study demonstrates a genotype-phenotype association that involves JAK2 and EPOR in the setting of PV, but not that of AMM. Some JAK2 SNPs were found to be associated with both PV and ET but in opposite direction. Therefore, genetic variation among MPDs might contribute to phenotypic diversity in the presence of specific mutations.

Abstract BACKGROUND. Several studies have recently reported on the occurrence of a JAK2 V617F mutation in myeloid cells from the majority of patients with polycythemia vera (PV). The clinical relevance of this novel observation is currently under study. Similarly, there is limited information regarding the correlation of JAK2 V617F mutational status and neutrophil PRV-1 transcript level in PV, secondary polycythemia (SP) and other myeloproliferative disorders (MPD). METHODS. In a single institutional study, mutation screening for JAK2 V617F was performed in DNA derived from archived blood granulocytes from 63 consecutive patients with PV in whom current diagnostic criteria were strictly applied and diagnosis confirmed by bone marrow histology. Concomitant quantitative analysis of neutrophil PRV-1 expression was performed in 28 of the 63 patients and the results of both JAK2 V617F and PRV-1 analysis in these 28 patients were compared with those from another 22 patients with essential thrombocythemia (ET), 10 with myelofibrosis with myeloid metaplasia (MMM), 19 with SP, and 11 healthy volunteers. RESULTS. The 63 patients with PV were followed for a median of 33 months (range 3–324) from the time of initial diagnosis. During this period, 3 patients have died including 2 from acute myeloid leukemia (AML). Disease transformation into either AML or MMM has occurred in 2 and 4 patients, respectively. The median time from diagnosis to the time of mutation analysis was 12 months (range 0–306). JAK2 V617F was detected in 58 of the 63 patients with PV (92%; homozygous in 21%). The clinical phenotype of the 5 patients with wild-type allele was otherwise typical for the disease. A statistical comparison between JAK2 V617F heterozygotes (n=45) and homozygotes (n=13) in PV did not reveal significant associations in regards to age, gender, leukocyte or platelet count at diagnosis, disease duration, or the incidences of thrombosis or bleeding. However, JAK2 V617F homozygotes, compared to their heterozygote counterparts, displayed significantly higher hemoglobin level at diagnosis (p=0.001), increased incidence of pruritus (69% vs. 38%; p=0.04), and a higher rate of fibrotic transformation (23% vs. 2%; p=0.009). The overall (homozygous) JAK2 V617F mutational frequencies in the other study patients with ET, MMM, SP, and healthy controls were 55% (0%), 30% (0%), 0%, and 0%, respectively. The corresponding figures for increased PRV-1 expression in these patients were 18%, 20%, 21%, and 9% as compared to 89% in PV. In patients with either ET or MMM, the likelihood of detecting JAK2 V617F was significantly higher in the presence of an increased PRV-1 expression (83% vs. 38%; p=0.05). Similarly, in patients with PV, homozygous as compared to heterozygous JAK2 V617F correlated with higher levels of PRV-1 expression (p=0.07). CONCLUSIONS. The current clinical study demonstrates a mutant allele dose effect on erythrocytosis, PV-characteristic clinical features, and granulocyte PRV-1 expression in PV. Furthermore, compared to the PRV-1 assay, mutation screening for JAK2 V617F displayed greater specificity in distinguishing PV from SP.

Abstract Background: In essential thrombocythemia (ET), homozygosity for the JAK2 V617F mutant allele is rare despite the frequent (23–57%) occurrence of the heterozygous state. This is in direct contrast with the situation in polycythemia vera (PV) where homozygosity for the mutation is seen in 25–33% of the cases. The particular observation is consistent with what has been observed in vitro as well as in animal studies where JAK2 V617F has been associated with induction of erythropoietin hypersensitivity in cell lines and erythrocytosis in mice (James et al. Nature2005;434:1144). Therefore, regardless of whether or not JAK2 V617F represents a primary event in myeloproliferative disorders, it is probable that it promotes a PV phenotype. In the current study, we explore this hypothesis as well as investigate the long-term prognostic relevance of the mutation in a well defined cohort of patients with ET seen at a single institution. Methods: The diagnosis of ET was according to the WHO classification of tumors (Jaffe ES et al. Lyon, IARC press, 2001). All patients were seen at the Mayo Clinic and patient characteristics and subsequent clinical events were determined both retrospectively (chart review) and prospectively (by means of a questionnaire sent both to patients and their primary doctors). In addition date and cause of death were confirmed by probing both the social security index and the national death index. DNA for mutation screening was derived and sequenced from either archived unsorted cells from the bone marrow or prospectively collected peripheral blood granulocytes according to previously published methods (Levine et al. Cancer Cell, 2005;7:387). Results: The study cohort consisted of 150 consecutive patients with ET including 99 females and 51 males (median age 49.8 years; range 16.8–98). Overall, JAK2 V617F was detected in 73 patients (48.7%) but none were homozygous for the mutant allele. At diagnosis, ET patients with JAK2 V617F were significantly older (median age of 53.5 vs. 42.3; p=0.02) and also displayed higher readings of both hemoglobin level (p=0.0002) and leukocyte count (p=0.005). All study patients have now been followed for a median of 11.4 years from diagnosis (range 0–32.7) and follow-up period was similar between the two mutational categories (p=0.53). During this period, 32 patients have died and the median survival for the entire group was 19.3 years; 21.9 and 17.7 years in the absence and presence of the mutation, respectively (p=0.09). Disease transformation into acute leukemia, myelofibrosis, and PV occurred in 6 (4%), 15 (10%), and 8 (5.3%) patients, respectively, and the only significant difference between the two mutational categories in this regard was the higher incidence of transformation into PV in patients with the mutation (p=0.02). Multivariate analysis identified age, hemoglobin level, and thrombosis history at diagnosis but not JAK2 V617F mutational status as independent risk factors for survival. Conclusion: Although the presence of JAK2 V617F in ET appears to promote a PV phenotype, it might not carry treatment-relevant information.

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

Abstract Background: An acquired point mutations of JAK2 (V617F) was recently described in a substantial proportion of patients with myeloproliferative disorders (MPD), including myelofibrosis with myeloid metaplasia (MMM). The current study examines i) JAK2 V617F mutational status at time of leukemic transformation (LT) of MMM and its clinical correlates, ii) the possibility of changes over time in mutational status in MMM patients that have either undergone leukemic transformation (LT) or experienced disease progression. Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from either peripheral blood mononuclear cells (PBMC) (i.e. fresh specimens) using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Fluorescent dye chemistry sequencing was performed using the same primers used for amplification. Results: The study population consisted of 68 patients including 33 patients with disease transformation into AML. i. MMM patient cohort with LT (n=33). The JAK2 V617F mutant allele was detected in 21 of the 33 patients (64%; homozygous in 6%) at the time of LT. As expected, PPMM was over-represented in the group with the mutation but the presence of the mutation affected neither the clinical presentation at time of LT or overall outcome. Furthermore, in 9 patients (7 heterozygotes and 2 with wild-type allele), archived bone marrow that was collected at a median of 23 months (range, 3–105) before LT was available and disclosed no change in mutational status. ii. MMM patient cohort with disease progression without LT (n=35; 19 AMM, 9 PPMM, 7 PTMM): Each patient in this group had at least two bone marrow or peripheral blood samples that were collected at two different time points separated by a median of 18.2 months (range (3–82). Of the total 35 patients in this group, 32 (91%) showed no change in JAK2 V617F mutational status over time including 3 patients in whom the first sample was collected before they transformed into MMM from antecedent PV or ET. In the remaining 3 patients, a switch from wild-type to heterozygous state (1 patient), and heterozygous to homozygous (2 patients), was documented after 78, 43, and 83 months, respectively. Conclusions: In most instances, JAK2 V617F mutational status in MMM remains unchanged over time including during leukemic transformation. Furthermore, the presence of the mutation in MMM-related LT does not appear to affect clinical outcome. These observations undermine the role of JAK2 V617F in either disease progression or LT in MMM.