Inhibition of cyclin‐dependent kinase 9 synergistically enhances venetoclax activity in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive and largely incurable subtype of non‐Hodgkin's lymphoma. Venetoclax has demonstrated efficacy in MCL patients with relapsed or refractory disease, however response is variable and less durable than CLL. This may be the result of co‐expression of other anti‐apoptotic proteins such as MCL‐1, which is associated with both intrinsic and acquired resistance to venetoclax in B‐cell malignancies. One strategy for neutralizing MCL‐1 and other short‐lived survival factors is to inhibit CDK9, which plays a key role in transcription. Here, we report the response of MCL cell lines and primary patient samples to the combination of venetoclax and novel CDK9 inhibitors. Primary samples represented de novo patients and relapsed disease, including relapse after ibrutinib failure. Despite the diverse responses to each single agent, possibly due to variable expression of the BCL‐2 family members, venetoclax plus CDK9 inhibitors synergistically induced apoptosis in MCL cells. The synergistic effect was also confirmed via venetoclax plus a direct MCL‐1 inhibitor. Murine xenograft studies demonstrated potent in vivo efficacy of venetoclax plus CDK9 inhibitor that was superior to each agent alone. Together, this study supports clinical investigation of this combination in MCL, including in patients who have progressed on ibrutinib

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T

Spliceosome Gene Mutations Are Frequently Found In JAK2 Negative Myelofibrosis and Associated With Worse Clinical Outcomes

Abstract Myelofibrosis (MF), a Philadelphia chromosome negative myeloproliferative neoplasm (MPN) requires clonal markers for accurate diagnosis according to the 2008 World Health Organization classification scheme. However, molecular mutations in Janus Kinase 2 (JAK2) are found in only 50% of MF patients (pts). Various somatic mutations identified in other myeloid cancers have also been found in MF with pre-existing JAK2 V617F mutations. We previously reported in 57 pts diagnosed with Triple Negative MF (TN-MF: JAK2 exon12/14 and wild type (WT) MPL) the absence of mutations in genes commonly identified in other myeloid malignancies, specifically TET2, DNMT3A, CBL, IDH1/2, SH3B2 (LNK), N/KRAS and EZH2. Only somatic mutations in ASXL1 were identified in a small cohort of TN-MF (16%). Mutations in components of the RNA splicing machinery have been recently identified in varying frequencies in different myeloid malignancies. We performed RNA-sequencing on 2 samples (JAK2 mutant (MUT) and 1 JAK2 WT) and found that 2 spliceosome genes (U2AF1 and SF3B1) had higher mRNA expression levels in the JAK2 WT (MFC=0.76 and 0.82, respectively) compared to JAK2 MUT patient. We hypothesized that somatic mutations in RNA splicing factor genes occur in TN-MF pts and may be helpful in the biological characterization of this group of pts. Therefore, we isolated DNA from bone marrow (BM) or peripheral blood mononuclear cells from a cohort of MF pts (N=132; JAK2 MUT:75 and JAK2 WT:57) and performed Sanger sequencing for SF3B1 (exons 13-16), U2AF1 (exons 2, 6 and 7), and SRSF2 (exons 1 and 2). Baseline characteristics of pts and clinical data including hematologic parameters, BM results, and presence of splenomegaly by palpation were collected. Pts were stratified based on the Dynamic International Prognostic Scoring System-Plus risk as high=40, Int-2=51, Int-1=11 and low-risk=5 pts. The mean duration of follow-up was 16.5 months in JAK2 MUT and 12.8 months in JAK2 WT. Spliceosome mutations were found in 35/132 (27%) (SRSF2=17%, U2AF1=8% and SF3B1=2%) of pts. Interestingly, the frequency of spliceosome mutations in JAK2 WT was higher (31%; SRSF2=26%, U2AF1= 5%, SF3B1=0) compared to JAK2 MUT (18%; SRSF2=10%, U2AF1=6%, SF3B1=2%) pts. Of note, the 2 pts with SF3B1 mutations (K700E) had ring sideroblast in the BM (occasional and 50%). A large number of WT JAK2 patients (50%) had concomitant SRSF2 and ASXL1 mutations. Both SRSF2 and ASXL1 have been associated with poor prognosis in MF. Indeed, in our JAK2 WT cohort, pts who harbored SRSF2 mutations had a higher mean percentage of BM blasts compared to the ones that were SRSF2 WT (5.6% vs 1.8%; P=.006). Moreover, JAK2 WT pts carrying spliceosome mutations had more severe anemia as shown by lower hemoglobin (8.98 g/dL vs 10.5) and higher leukocyte counts (27.5 x109/L vs 20.5) compared to WT cases. Furthermore, we noticed that they also had higher frequency of RBC (Red Blood Cells) transfusions compared to WT (60% vs 40%) cases. Spleen examination by palpation below the left sub-costal margin showed that JAK2 WT cases carrying spliceosome mutations had larger splenomegaly compared to WT cases [10.1 cm (range 4-26), vs 8.7cm (range 2-20); P=0.05]. In conclusion, molecular alterations in the spliceosome machinery are frequently found in MF pts who are WT for JAK2/MPL and are associated with higher BM blast percentage, more severe anemia, higher leukocyte counts, transfusion dependence and more prominent splenomegaly. The observation of appreciable frequency of spliceosome mutations in JAK2 WT MF pts opens the possibility of using spliceosome inhibitors in the management of this disease group. Disclosures: No relevant conflicts of interest to declare

Triple Negative (JAK2 exon 12 /14 and MPL wild type) Myelofibrosis Have Higher Expression Of CDC25A and Greater Sensitivity To CDC25A Inhibition Compared To JAK2 Mutant Cases

Abstract Pharmacologic therapies that target the JAK-STAT pathway are clinically used to alleviate splenomegaly and disease-related constitutional symptoms in MF. However, it is clear that some patients develop intolerance or resistant to this therapy. Furthermore, there are MF related complications especially cytopenias that are not alleviated by these therapies. Therefore, alternative and complementarytherapies are warranted in the management of MF. We hypothesized that other pathways downstream of the JAK-STAT signaling pathway can play a role in the pathophysiology of MF. We used whole exome (WES) and RNA sequencing technologies to interrogate new molecular markers and pathways which can serve as novel targets for this disease. In 4 MF patients [JAK2 mutant (MUT) =2, and wild type (WT) =2], WES was performed using the Illumina platform. All of the variants were filtered based on PHRED score (>=30) with coverage was set at 30X. Analysis of data in JAK2/MPL WT patients demonstrated the presence of 263 candidate genes. After clarifying the status of tumor nucleotide variants in each gene compared to germline (CD3+) fraction, 7 genes (RBL1, ADSS, ZNF717,MUC4, TUBB4Q and CDC25A) were selected for further somatic confirmation by direct sequencing. Among these genes, only alteration in CDC25A, a regulator of cyclinE/cdk2 (cyclin-dependent kinase-2) and cyclinA/cdk2 kinase, was confirmed to be somatic. This genetic change was previously reported as somatic by WES in lung cancer although not confirmed by direct sequencing (Bartkova et al, Nature, 2005, Apr 14; 434 (7035):864-70). Based on these observations and since CDC25A acts as a downstream effector of JAK-STAT signaling, we hypothesized that, CDC25A phosphatase, may be a driver in MF pathogenesis. The transcriptome of two patients, one MUT and one WT for JAK2 was then analyzed. RNA was isolated from bone marrow (BM) cells of healthy individuals (HI) (N=3). cDNA was made from 1.5-3 ug of RNA and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads. Paired-end 90 base pair reads were generated on an Illumina HiSeq2000 sequencer and aligned to the human genome 19. RNA-splicing patterns were analyzed by a bioinformatics algorithm and gene expression analysis was carried out using GSEA (Visconte V; Blood. 2012). By using FDR80%) while JAK2 MUT samples had only a few positive megakaryocytes (<20%). To test the feasibility of targeting this pathway in patients with MF and to assess for differential response between JAK2 MUT and WT cases, a potent cell permeable 7-substituted quinolinedione derivedCDC25 phosphatase inhibitor (NSC663284) was tested in JAK2 MUT (N=2) vs WT (N=1). Cell proliferation was determined by Trypan Blue and MTT assay after cell exposure to different concentrations of the inhibitor [3, 5, 7, 10 and 30uM] in 24 hours observation. NSC663284 induced higher dose-dependent cell growth inhibition in JAK2 WT compared to MUT cases (% of viable cells in WT vs MUT using previously mentioned concentrations, 3 uM= 98% vs 86%, 5 uM= 93% VS 77%, 7 uM= 88% vs 65%, 10 uM= 71% vs 43% and 30 uM= 25% vs 61%; p=0.01).In conclusion, CDC25A is more highly expressed in patients with wild type JAK2 compared to the mutant counterpart and primary cells from WT JAK2 patients demonstrate higher sensitivity to CDC25A inhibition, warranting further clinical testing of this therapeutic strategy. Disclosures: No relevant conflicts of interest to declare