SF3B1 Mutations Research Articles

Many faces of SF3B1-mutated myeloid neoplasms: concurrent mutational profiles contribute to the diverse clinical and morphologic features

Splicing factor SF3B1 mutation occurs in 20-30% of myelodysplastic syndrome (MDS) and myelodysplasia/myeloproliferative neoplasm (MDS/MPN), particularly those with ring sideroblasts (RS), and rarely in acute myeloid leukemia (AML). In this study, we performed a comprehensive evaluation of 77 SF3B1-mutated myeloid neoplasms (45 MDS, 18 MDS/MPN, 13 AML, and 1 MPN), including their clinical presentations, morphologic features, cytogenetic studies, and targeted next-generation sequencing. Our study demonstrated that concurrent gene mutations were very different in SF3B1-mutated MDS, MDS/MPN, and AML. MDS cases were frequently characterized by either sole SF3B1 mutation or in combination with TET2 mutation. Acquiring additional mutations in transcription factors, such as RUNX1 and GATA2, were associated with increased blasts and progression to AML in patients with MDS or MDS/MPN. Our study also demonstrated that SF3B1-mutated MDS/MPN was not only associated with thrombocytosis (5/18, 27.7%), defined by the current WHO classification as MDS/MPN-RS-T, but also associated with neutrophilia (6/18, 33.3%), monocytosis (6/18, 33.3%), and mastocytosis (1/18, 5.6%). Our results indicate that although SF3B1-mutated myeloid neoplasms in general have a good prognosis, evaluation of the concurrent gene mutational profile is important for risk stratification. In addition, our study, in combination with other published data, suggests that the category of MDS/MPN-RS-T in the current WHO classification could be expanded to include SF3B1-mutated MDS/MPN-RS with peripheral leukocytosis such as neutrophilia and monocytosis.

Evaluation of the prognostic significance of SF3B1 mutation in Iraqi patients with chronic lymphocytic leukemia (CLL)

Evaluation of the prognostic significance of SF3B1 mutation in Iraqi patients with chronic lymphocytic leukemia (CLL)

Abstract IA26: Understanding CLL and Richter’s syndrome biology through mouse models of human genetics

Abstract Large-scale human next generation sequencing studies have progressively defined the complex genetic landscape of chronic lymphocytic leukemia (CLL) and Richter’s syndrome (RS), yet the difficulties in rapid manipulation of primary CLL cells and the limited availability of cell lines have challenged studies aimed at interrogating the function of the so-called disease “drivers”, defined in human studies through statistical inference. Mouse models represent powerful tools to study mechanisms of normal and malignant B cell biology. Conditional knock-in/knock-out systems have allowed B-cell restricted modeling of CLL genetic lesions, with MDR mice being the first example of a genetically-faithful CLL model, recapitulating the most common genomic aberration of human disease, del(13q). Since then, a series of models generated by our group and others have allowed to uncover key functional changes underlying disease onset and transformation, including in the context of combined mutation in the splicing factor Sf3b1 and the DNA damage response gene Atm, sole mutation in the B cell developmental factor Ikzf3, the nuclear export receptor Xpo1, the ribosomal protein Rps15, and the DNA methyltransferase Dnmt3a (the latter not a CLL lesion per se, but recapitulating disordered methylation patterns typical of CLL). The CRISPR-Cas9 gene editing technology has further enabled B-cell restricted introduction of multiple combinatorial loss-of-function lesions, allowing the generation of faithful models not only of CLL but, importantly, of transformation into RS. In this presentation, I will describe the application of mouse models to studies of CLL and RS pathogenesis and treatment, with a focus on key aspects uncovered through the characterization of genetically- inspired mouse lines of recurrent disease drivers. I will provide a perspective on how these novel models combined with new technology allowed the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible, representing valuable platforms for functional genomic analyses. Citation Format: Elisa ten Hacken. Understanding CLL and Richter’s syndrome biology through mouse models of human genetics [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr IA26.

Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations.

Mutations in the SF3B1 splicing factor are commonly seen in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), yet the specific oncogenic pathways activated by mis-splicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in the pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. RNA-seq data from SF3B1 mutant samples was analyzed and functional roles of interleukin-1 receptor-associated kinase 4 (IRAK4) isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in preclinical models of MDS/AML. RNA-seq splicing analysis of SF3B1 mutant MDS samples revealed retention of full-length exon 6 of IRAK4, a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteasomal degradation. Expression of IRAK4-long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. SF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models. This work was supported by Cincinnati Children's Hospital Research Foundation, Leukemia Lymphoma Society, and National Institute of Health (R35HL135787, RO1HL111103, RO1DK102759, RO1HL114582), Gabrielle's Angel Foundation for Cancer Research, and Edward P. Evans Foundation grants to DTS. AV is supported by Edward P. Evans Foundation, National Institute of Health (R01HL150832, R01HL139487, R01CA275007), Leukemia and Lymphoma Society, Curis and a gift from the Jane and Myles P. Dempsey family. AP and JB are supported by Blood Cancer UK (grants 13042 and 19004). GC is supported by a training grant from NYSTEM. We acknowledge support of this research from The Einstein Training Program in Stem Cell Research from the Empire State Stem Cell Fund through New York State Department of Health Contract C34874GG. MS is supported by a National Institute of Health Research Training and Career Development Grant (F31HL132420).

Correlation between BAP1 Localization, Driver Mutations, and Patient Survival in Uveal Melanoma.

Simple Summary100 uveal melanomas from the UK were analyzed for molecular biomarkers, including alterations of chromosomes 3 and 8, cellular localization of BAP1, and genes known to be mutated in uveal melanoma. Consistent with earlier studies, loss of nuclear BAP1 (nBAP1) predicted shorter overall survival. Tumors with BAP1 loss of function mutations frequently exhibited heterogeneous BAP1 staining, and tumors with ≥25% loss of nBAP1 were a more reliable prognosistic indicator than chromosome 3 loss (LOH3) or chromosome 8q gain. Regardless of mutation class, most BAP1 mutations led to loss of nBAP1 and aberrant expression of cBAP1.Uveal melanoma (UM) is an uncommon but highly aggressive ocular malignancy. Poor overall survival is associated with deleterious BAP1 alterations, which frequently occur with monosomy 3 (LOH3) and a characteristic gene expression profile. Tumor DNA from a cohort of 100 UM patients from Moorfields Biobank (UK) that had undergone enucleation were sequenced for known UM driver genes (BAP1, SF3B1, EIF1AX, GNAQ, and GNA11). Immunohistochemical staining of BAP1 and interphase FISH for chromosomes 3 and 8 was performed, and cellular localization of BAP1 was correlated with BAP1 mutations. Wildtype (WT) BAP1 staining was characterized by nBAP1 expression with <10% cytoplasmic BAP1 (cBAP1). Tumors exhibited heterogeneity with respect to BAP1 staining with different percentages of nBAP1 loss: ≥25% loss of nuclear BAP1 (nBAP1) was superior to chr8q and LOH3 as a prognostic indicator. Of the successfully sequenced UMs, 38% harbored oncogenic mutations in GNA11 and 48% harbored mutations in GNAQ at residues 209 or 183. Of the secondary drivers, 39% of mutations were in BAP1, 11% were in EIF1AX, and 20% were in the SF3B1 R625 hotspot. Most tumors with SF3B1 or EIF1AX mutations retained nuclear BAP1 (nBAP1). The majority of tumor samples with likely pathogenic BAP1 mutations, regardless of mutation class, displayed ≥25% loss of nBAP1. This included all tumors with truncating mutations and 80% of tumors with missense mutations. In addition, 60% of tumors with truncating mutations and 82% of tumors with missense mutations expressed >10% cBAP1.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

SF3B1 facilitates HIF1-signaling and promotes malignancy in pancreatic cancer.

Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors.

Update in Pathogenesis, Diagnosis, and Therapy of Prolactinoma.

Simple SummaryThis review updates recent advances in the pathogenesis, diagnosis, and therapy of prolactinoma. Prolactinomas, comprising 30–50% of all pituitary neuroendocrine tumors, frequently occur in females aged 20 to 50 and cause hypogonadism and infertility. In typical cases, female patients exhibit galactorrhea and amenorrhea due to serum prolactin (PRL) elevation, and during pregnancy, they should be carefully treated. During diagnosis, other causes of hyperprolactinemia must be excluded, and an MRI is useful for detecting pituitary neuroendocrine tumors. For the treatment of prolactinoma, dopamine agonists are effective in decreasing PRL levels and shrinking tumor size in most patients. Surgical treatment is recommended for patients who are resistant or intolerant to dopamine agonists. This review also discusses giant and malignant prolactinomas, prolactinoma-associated pregnancy, and new therapeutic approaches.Prolactinomas comprise 30–50% of all pituitary neuroendocrine tumors, frequently occur in females aged 20 to 50, and cause hypogonadism and infertility. In typical cases, female patients exhibit galactorrhea and amenorrhea due to serum prolactin (PRL) elevation, and patients during pregnancy should be carefully treated. During diagnosis, other causes of hyperprolactinemia must be excluded, and an MRI is useful for detecting pituitary neuroendocrine tumors. For treating prolactinoma, dopamine agonists (DAs) are effective for decreasing PRL levels and shrinking tumor size in most patients. Some DA-resistant cases and the molecular mechanisms of resistance to a DA are partially clarified. The side effects of a DA include cardiac valve alterations and impulse control disorders. Although surgical therapies are invasive, recent analysis shows that long-term remission rates are higher than from medical therapies. The treatments for giant or malignant prolactinomas are challenging, and the combination of medication, surgery, and radiation therapy should be considered. Regarding pathogenesis, somatic SF3B1 mutations were recently identified even though molecular mechanisms in most cases of prolactinoma have not been elucidated. To understand the pathogenesis of prolactinomas, the development of new therapeutic approaches for treatment-resistant patients is expected. This review updates the recent advances in understanding the pathogenesis, diagnosis, and therapy of prolactinoma.

Cytogenetic and Genetic Abnormalities with Diagnostic Value in Myelodysplastic Syndromes (MDS): Focus on the Pre-Messenger RNA Splicing Process.

Myelodysplastic syndromes (MDS) are considered to be diseases associated with splicing defects. A large number of genes involved in the pre-messenger RNA splicing process are mutated in MDS. Deletion of 5q and 7q are of diagnostic value, and those chromosome regions bear the numbers of splicing genes potentially deleted in del(5q) and del(7q)/-7 MDS. In this review, we present the splicing genes already known or suspected to be implicated in MDS pathogenesis. First, we focus on the splicing genes located on chromosome 5 (HNRNPA0, RBM27, RBM22, SLU7, DDX41), chromosome 7 (LUC7L2), and on the SF3B1 gene since both chromosome aberrations and the SF3B1 mutation are the only genetic abnormalities in splicing genes with clear diagnostic values. Then, we present and discuss other splicing genes that are showing a prognostic interest (SRSF2, U2AF1, ZRSR2, U2AF2, and PRPF8). Finally, we discuss the haploinsufficiency of splicing genes, especially from chromosomes 5 and 7, the important amplifier process of splicing defects, and the cumulative and synergistic effect of splicing genes defects in the MDS pathogenesis. At the time, when many authors suggest including the sequencing of some splicing genes to improve the diagnosis and the prognosis of MDS, a better understanding of these cooperative defects is needed.

Genetic mutations associated with blood count abnormalities in myeloid neoplasms

ABSTRACT Introduction Myelodysplastic syndromes (MDS) predominantly present with varying degrees of cytopenia, while myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) exhibit proliferative features. Genetic defects underlying different complete blood count (CBC) alterations remain to be defined. Objective We aimed to evaluate mutations and impacts on abnormal blood counts in MDS and MDS/MPN. Method MDS and MDS/MPN patients were recruited and sequenced by targeted next-generation sequencing. Clinical parameters, especially CBC, were evaluated for the association with genetic abnormalities and clinical outcomes. Results A total of 168 patients with myeloid neoplasms were recruited (92 cases of low-risk MDS, 57 cases of high-risk MDS and 19 cases of MDS/MPN). Compared to low-risk MDS and MDS/MPN, patients with high-risk MDS were presented with more severe neutropenia with 17.5% showing absolute neutrophil counts (ANC) lower than 0.5 × 109/L. Patients with MDS/MPN more commonly harboured mutations and had a higher number of mutations per case than low-risk MDS (94.7% vs. 56.5%; p < 0.001 and 3 vs. 1; p < 0.001, respectively). Patients with SF3B1 mutations showed lower haemoglobin levels than wild-type (7.9 vs. 8.4 g/dL, p = 0.02), but were associated with normal platelet counts (286 vs. 93 × 109/L; p < 0.001). Patients with U2AF1 mutations were associated with more severe leukopenia than wild-type (3 vs. 4.18 × 109/L; p = 0.02). KRAS mutations were associated with monocytosis (p < 0.001). Multivariate analysis revealed high-risk MDS, MDS/MPN, severe neutropenia (ANC < 0.5 × 109/L), and mutations in ASXL1 and SETBP1 were associated with inferior survival outcomes. Conclusion Certain mutations were related to more severe anaemia, lower white blood cell count or monocytosis in Asian MDS and MDS/MPN patients.

A Novel Prognostic Scoring Model for Myelodysplastic Syndrome Patients With SF3B1 Mutation.

The outcomes of myelodysplastic syndrome (MDS) patients with SF3B1 mutation, despite identified as a favorable prognostic biomarker, are variable. To comprehend the heterogeneity in clinical characteristics and outcomes, we reviewed 140 MDS patients with SF3B1 mutation in Zhejiang province of China. Seventy-three (52.1%) patients diagnosed as MDS with ring sideroblasts (MDS-RS) following the 2016 World Health Organization (WHO) classification and 118 (84.3%) patients belonged to lower risk following the revised International Prognostic Scoring System (IPSS-R). Although clonal hematopoiesis-associated mutations containing TET2, ASXL1 and DNMT3A were the most frequent co-mutant genes in these patients, RUNX1, EZH2, NF1 and KRAS/NRAS mutations had significant effects on overall survival (OS). Based on that we developed a risk scoring model as IPSS-R×0.4+RUNX1×1.1+EZH2×0.6+RAS×0.9+NF1×1.6. Patients were categorized into two subgroups: low-risk (L-R, score <= 1.4) group and high risk (H-R, score > 1.4) group. The 3-year OS for the L-R and H-R groups was 91.88% (95% CI, 83.27%-100%) and 38.14% (95% CI, 24.08%-60.40%), respectively (P<0.001). This proposed model distinctly outperformed the widely used IPSS-R. In summary, we constructed and validated a personalized prediction model of MDS patients with SF3B1 mutation that can better predict the survival of these patients.

A novel SF3B1 variant in a patient with MDS with ring sideroblasts.

An 80-year-old male was investigated for anemia. Complete blood counts showed WBC of 4.0 × 109/L, hemoglobin of 92 g/L, hematocrit of 27.2%, mean corpuscular volume of 103.7 fl, red cell distribution width of 19.7%, and platelets of 140 × 109/L. Aspirate smears showed 4% blasts, decreased myeloid to erythroid ratio (0.7:1), and megaloblastoid erythroid elements (>10%; Figure 1, panel A, original magnification 1000×, Wright–Giemsa stain). The iron stain showed ∼94% ring sideroblasts (Figure 1, panel B, original magnification 1000×, iron stain). The bone marrow biopsy showed a 60% cellular marrow with occasional hypolobulated megakaryocytes (>10%; Figure 1, panel C, original magnification 200×, hematoxylin and eosin stain; Figure 1, panel D, original magnification 400×, Wright–Giemsa stain). A custom next-generation sequencing panel (45 genes, RainDance/Illumina) showed SF3B1 c.1862A>T, p.D621V at 39.9% variant allele frequency (VAF) and TET2 c.1862A>T, p.S1898F at 3.6% VAF. Cytogenetics showed a normal karyotype. The diagnosis of myelodysplastic syndrome (MDS) with ring sideroblasts (RS) and multilineage dysplasia (MLD) was rendered. To our knowledge, SF3B1 c.1862A>T, p.D621V identified in this case has never been reported to be pathogenic. This case highlights how the morphologic findings (i.e., ring sideroblasts) were very important to guide the interpretation of this novel SF3B1 variant as pathogenic. And, in turn, the molecular evidence of an SF3B1 mutation permitted definitive diagnosis and classification of the morphologic findings. The authors declare no conflict of interest. This study received no specific funding. The authors agree for data sharing and the journal policy.

P735: THE ROLE OF SF3B1 MUTATIONS IN MYELODYSPLASTIC SYNDROMES

Background:SF3B1 is one of the most commonly mutated genes in patients with myelodyplastic syndromes (MDS), associated with ring sideroblasts (RS) and an indolent disease course. Malcovati et al. proposed MDS with mutated SF3B1 fulfilling certain criteria as distinct MDS entity (Malcovati et al., Blood, 2020). Aims: Study the SF3B1 mutation in MDS with respect to the genomic landscape, AML transformation rate and clinical outcome. Methods: We analyzed 734 patients diagnosed with MDS based on cytomorphology, cytogenetics and molecular genetics and classified according to WHO classification 2017. Amplification-free WGS was performed with a median coverage of 103x (Illumina, San Diego, CA). Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7. Results:SF3B1 mutations were identified in 231 of 734 (31%) MDS patients and were mainly found in MDS-RS (171/200; 86%; MDS-RS-SLD: 43/51, 84%; MDS-RS-MLD: 128/149; 86%). In addition, 13% (37/300) of MDS with excess blasts (MDS-EB1/2) and 20% of MDS with isolated del(5q) harbored SF3B1 mutations. In the total cohort SF3B1 mutations were associated with better outcome (median OS: 79 vs. 53 month; p<0.0001). Within the different MDS entities SF3B1 mutations were favorable in MDS-RS-SLD (median OS: 106 vs. 25 month; p=0.009), MDS-RS-MLD (median OS: 82 vs. 64 month; p=0.049) and MDS-EB-2 (median OS: 129 vs. 25 month; p=0.011), but were associated with shorter survival in MDS with isolated del(5q) (median OS: 69 vs. 79 month; p=0.044). 144/231 (62%) SF3B1 mutated samples fulfilled the criteria proposed by Malcovati et al. (SF3B1ent). These cases were associated with longer survival compared to SF3B1 mutated samples not falling into the proposed SF3B1 entity (SF3B1nent). Within SF3B1ent 47% (67/144) did not harbor any additional mutation resulting in an average of 1.8 mutations (including SF3B1) in this group. SF3B1nent patients showed on average more mutations (MDS with isolated del(5q): 1.9; MDS-EB: 2.7; MDS-RS: 3.1). The most frequent additional mutations in all SF3B1 mutated patients were TET2 (29%), DNMT3A (16%) and ASXL1 (9%). Regarding cytogenetic abnormalities, 69/231 (30%) SF3B1 mutated samples showed aberrant karyotypes (SF3B1ent: 27/144, 19%; SF3B1nent: 42/87, 48%). Of SF3B1 mutated patients 7% (15/231) progressed to AML compared to 15% (75/503) of SF3B1 wildtype patients (median follow-up: 112 months). Notably, 80% of SF3B1nent showed AML transformations in comparison to 20% of SF3B1ent (median follow-up: 122 and 112 months). Within the 15 SF3B1 mutated patients progressing to AML 47% showed additional RUNX1 and 27% DNMT3A or TET2 mutations at MDS diagnosis. In AML-transforming patients with a low SF3B1 variant allelic frequency (VAF, n=2) additional spliceosome mutations (SRSF2 or ZRSF2) were identified at the time of diagnosis. Two other SF3B1 mutated patients showing disease progression to AML harbored MECOM rearrangements when MDS was diagnosed. During progression one patient gained a MECOM rearrangement and two patients other chromosomal aberrations present in the AML state. Image:Summary/Conclusion:SF3B1 mutations occur in several MDS entities, are associated with good clinical outcome and mainly co-occurred with TET2 mutations. Patients showed a better prognosis (longer OS, lower AML transformation rate) when categorized into the proposed SF3B1 entity. AML transformation of SF3B1 mutated MDS patients is determined by the entire genomic landscape indicating RUNX1 as potential driver.

P411: SF3B1 MUTATIONS IN AML ARE STRONGLY ASSOCIATED WITH MECOM REARRANGEMENTS AND MAY BE INDICATIVE OF AN MDS PRE-PHASE

Background: Mutations in the splicing factor gene SF3B1 are most frequent in myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) but are also recurrently found in acute myeloid leukemia (AML). Splicing factor mutations have been suggested to have additive value if incorporated into the current risk classification of AML (van der Werf et al., Blood Advances 2021). Aims: Explore the genetic landscape of SF3B1 mutated AML and determine the prognostic impact of SF3B1 mutations. Methods: We analyzed 735 patients diagnosed with AML based on cytomorphology, cytogenetics, and molecular genetics according to WHO classification 2017. Reads from amplification-free whole genome sequencing (WGS, median coverage 103x, Illumina, San Diego, CA) were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7. Results: We identified SF3B1 mutations in 41 of 735 (6%) AML patients. SF3B1 mutations were found in AML with recurrent genetic abnormalities (24/471; 5%), AML with myelodysplasia-related changes (MRC; 11/158; 7%) and AML not otherwise specified (NOS; 6/106; 6%). When focussing on SF3B1mut cases, AML-MRC (11/41; 27%) and AML with GATA2::MECOM (10/41; 24%) were most frequent. 9 additional cases showed another MECOM rearrangement (-rear) resulting in 46% (19/41) of patients with SF3B1 mutations accompanied by a MECOM-rear. A prior history of MDS or MDS/MPN was documented in 20% (8/41) of SF3B1mut patients. Thereof, 63% (5/8) harbored a MECOM rearrangement and 25% RUNX1mut. On average, SF3B1mut patients harbored 2.85 mutations (including SF3B1). The most frequent additional mutations of SF3B1mut patients were RUNX1 (9/41; 22%) and NRAS (8/41; 20%), and NPM1, TET2, or DNMT3A mutations or FLT3-ITD were detected in 15% (6/41) each. SF3B1 showed an average variant allelic frequency (VAF) of 41%. Notably, the VAF of SF3B1 in AML with NPM1 or RUNX1 was higher than the VAFs of NPM1 or RUNX1 in 9/11 (82%) cases, suggesting that SF3B1 preceded these mutations. In 16/41 (39%) cases molecular follow-up data was available. The SF3B1 mutation persisted during the entire disease course in 8 cases (including 1 with relapse). In particular, in two of these AML patients with mutated NPM1, the SF3B1 mutation remained detectable by NGS, despite complete hematologic remission and undetectable NPM1 mutation by high-sensitive qPCR. In all 5 cases with relapse the SF3B1 mutation re-occurred at relapse. In 4 patients, the VAF of SF3B1 decreased, similar to accompanying mutations. In contrast to MDS, SF3B1 mutations did not affect the overall survival (median OS: 16.9 months in SF3B1mut; 5.7 months in unmutated group; p=0.822) in the total AML cohort. When stratified for AML sub-entities, the prognosis of the SF3B1mut AML seems to be dominated by the sub-entity. Image:Summary/Conclusion: Within AML, SF3B1 mutations are enriched in AML patients with GATA2::MECOM and AML-MRC. This association with poor risk subtypes dominates the prognosis of SF3B1mut AML. The high VAF of SF3B1 mutations and the persistence of SF3B1 mutations in AML patients in complete remission suggest the early acquisition of SF3B1 mutations in a pre-leukemic clone in a number of patients. While an antecedent MDS or MDS/MPN has been documented in some cases of SF3B1mut AML, it might be unidentifiable in others. Alternatively, SF3B1mut clonal hematopoiesis of indeterminate potential (CHIP) may represent a relevant pre-phase of SF3B1mut AML.

S129: TAKEAIM LEUKEMIA- A PHASE 1/2A STUDY OF THE IRAK4 INHIBITOR EMAVUSERTIB (CA-4948) AS MONOTHERAPY OR IN COMBINATION WITH AZACITIDINE OR VENETOCLAX IN RELAPSED/REFRACTORY AML OR MDS

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) and FLT3. IRAK4 is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of IRAK4 and have been associated with disease progression and poor prognosis of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML). Aims: Assessment of safety, clinical activity, and Recommended Phase 2 Dose (RP2D) of emavusertib as monotherapy or in combination with azacitidine (AZA) or venetoclax (VEN). Methods: This is an open-label, phase 1/2a dose escalation and cohort expansion trial (NCT04278768). In phase 1 Dose Escalation, patients with R/R AML or HR-MDS are treated with emavusertib monotherapy. Phase 1b includes 2 arms of combination therapy: emavusertib + AZA and emavusertib + VEN. The primary objectives of this study are to assess the safety, clinical activity, and identify the RP2D of emavusertib as monotherapy or in combination with AZA or VEN in R/R AML or HR-MDS. The Phase 2a Dose Expansion includes patients for emavusertib monotherapy: R/R AML with FLT3 mutation, or AML and HR-MDS R/R to HMA with U2AF1 or SF3B1 mutations. Results: As of December 16th, 2021, 49 patients have been treated in the phase 1 portion, of whom 43 started by September 30th, allowing 2 on-study disease assessments. The median number of prior therapies was 2 (range 1-5). Four monotherapy dose levels of emavusertib were tested (200 to 500 mg orally BID). No dose-limiting toxicities were observed at 200 mg and 300 mg BID. No Grade 4 or 5 treatment-related AEs (TRAEs) were reported, and all the TRAEs were manageable. Reversible, manageable Grade 3 rhabdomyolysis occurred in 1/26 (4%) patients at 300 mg BID, 2/17 (12%) at 400 mg BID, and 1/3 (33%) at 500 mg BID. RP2D was determined as 300 mg BID. Of 43 patients starting before Sept 30th, 2021, 14 had SF3B1, U2AF1 or FLT3 mutations and demonstrated more promising efficacy. In the 5 evaluable AML patients with spliceosome mutations, 40% reached CR/CRh (1 CR, 1 CRh), both with study duration >6 months. In the 7 spliceosome-mutated HR-MDS patients, 57% reached marrow CR, including 1 with RBC transfusion independence and 1 proceeding to HSCT. One of the three FLT3-mutated AML reached CR, and 2 became FLT3-negative. Among the 29 patients without SF3B1/U2AF1/FLT3 mutations, 1 reached CR and 2 PR. Phase 1b and Phase 2a are ongoing. RNA-seq on selected samples showed decrease in relative expression of IRAK4-long isoforms with response to emavusertib. Summary/Conclusion: Emavusertib is well tolerated and effective in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1/SF3B1/FLT3 mutations. No dose-limiting myelosuppression was reported, suggesting emavusertib may be a candidate for combination therapy. Accrual of Phases 1b and 2a is ongoing.

P388: PREFERENTIAL SYNERGISTIC EFFECT OF INDISULAM IN COMBINATION WITH VENETOCLAX AND AZACYTIDINE IN AML CELLS WITH SF3B1 MUTATIONS.

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis characterized by a high rate of relapse after conventional combination chemotherapy, highlighting the need for improved therapeutic interventions. Genes involved in pre-mRNA splicing, namely SF3B1, U2AF1 and SRSF2, are frequently mutated in AML.Recent studies suggest that pharmacological targeting of the splicing machinery results in preferential lethality of splicing mutant AML cells, potentially providing a novel strategy for treatment of this disease. Existing anti- cancer sulfonamides have been shown to interfere with splicing and induce preferential killing of splicing mutant AML cell lines. A particular sulfonamide drug, indisulam, was previously studied in cancer clinical trials where it was found to be safe but demonstrated limited efficacy, most likely because neither the mechanism of action nor potential biomarkers of response were known. Aims: We investigated, in this study, the efficacy of indisulam, either alone or in combination with ABT-199 (venetoclax) and azacitidine, in cultured primary human AML cells. Methods: Bone marrow cells were collected from adults newly diagnosed with AML, non-treated. Cells were cultured and incubated for 48 and 72 hours with indisulam at a range of nanomolar concentrations. Additionally, cells were incubated with ABT-199 (10nM) and azacitidine (1μM), alone or in combination with indisulam. Cell viability and cell death were measured. For mutation profiling, a targeted myeloid gene panel was used. Results: We found that treatment with indisulam resulted in lethality of primary AML cells harboring mutations in SF3B1, but had minor or no effect on AML cells with other mutations including mutations in splicing factors U2AF1 and SRSF2. In SF3B1-mutated cells, a clear synergistic effect was further observed when indisulam was combined with ABT-199 and azacitidine. The correlation between efficacy of indisulam and synergy with ABT-199 and azacitidine in AML cells with different mutations will be shown and discussed. Summary/Conclusion: Our results indicate that indisulam is preferentially active and synergistic with venetoclax and azacitidine in SF3B1-mutated cells from newly diagnosed, non-treated AML patients.

P783: SYSTEMIC THERAPY UTILIZATION AND HEMATOLOGIC OUTCOMES IN LOWER-RISK MYELODYSPLASTIC SYNDROMES (LR-MDS): FINDINGS FROM A REAL-WORLD MEDICAL RECORD REVIEW STUDY IN THE US, UK, AND EUROPE (EU)

Background: Patients (pts) with LR-MDS experience a mean life-year loss of 6 years, and the treatment focus is improvement of quality of life and prolongation of life expectancy. Aims: This retrospective medical record review assessed real-world data to determine prevailing LR-MDS treatment patterns and hematologic outcomes. Methods: Eligible pts (≥18 years of age) with LR-MDS (Jul 1, 2013–Sep 30, 2018) were identified by participating hematologist-oncologists in the US, Canada (CAN), UK, and EU (France, Germany, Spain). Study measures and hematologic outcomes were descriptively assessed (data abstraction Jun–Nov 2021). Aggregated analyses used pooled US/CAN and UK/EU data sets for the overall population and pt subgroups treated with clinician-defined first-line (1L) and second-line (2L) erythropoiesis-stimulating agents (ESAs). Results: Medical record data were abstracted by 114 US/CAN (n=174 pts; median age 60.6 years; 68.4% male) and 166 UK/EU clinicians (n=319 pts; median age 67.1 years; 72.4% male). Median follow-up duration from MDS diagnosis was 60.6 months (US/CAN) and 67.6 months (UK/EU). Approximately half of the pts had “low” risk status (49.4% US/CAN; 53.9% UK/EU) categorized by the Revised International Prognostic Scoring System at initial diagnosis. The most common karyotype abnormality was del(5q) (27.6% US/CAN; 20.4% UK/EU). SF3B1 mutation was recorded for 7.5% US/CAN and 8.2% UK/EU pts. Almost all pts had received ≥1 line of systemic treatment for managing MDS-associated anemia (US/CAN: 165 [94.8%] 1L, 77 [44.3%] 2L; UK/EU: 294 [92.2%] 1L, 133 [41.7%] 2L). In 1L, ESA-based regimens were the most common treatment with median duration of therapy of ≥2 years (US/CAN: 142 [86.1%], of which the majority (80.3%) was ESA monotherapy; UK/EU: 264 [89.8%], comprising 82.2% ESA monotherapy). Treatment characteristics and outcomes are shown in the Table. Among pts who were transfusion dependent (TD) before 1L ESA, a very small proportion achieved transfusion independence (TI) or red blood cell transfusion reduction ≥50%, which was maintained for a median of <1.5 years. Among the 1L ESA-treated pts who were TD, TI was achieved by 5.7% (US/CAN) and 12.9% (UK/EU). In 2L ESA-treated pts (n=23 US/CAN; n=46 UK/EU), almost all were previously treated with a 1L ESA-containing regimen (95.7% US/CAN; 84.8% UK/EU). The most common reasons for reinitiating ESA in 2L (n=22, 15.5% US/CAN; n=39, 14.8% UK/EU) were overall health status (40.9% US/CAN; 33.3% UK/EU), treatment efficacy (40.9% US/CAN; 48.7% UK/EU), and compliance with national guidelines (13.6% US/CAN; 46.2% UK/EU). Among the pts who reinitiated ESA in 2L, the most common reasons for discontinuation of 1L ESA were completion of planned course of therapy (45.5% US/CAN; 28.2% UK/EU), pt decision (40.9% US/CAN; 20.5% UK/EU), progressive disease (36.4% US/CAN; 51.3% UK/EU), and adverse events (13.4% US/CAN; 15.4% UK/EU). Lenalidomide was used in 1L therapy among 8.5% pts in US/CAN and 5.8% in UK/EU. Among those with del(5q) mutation, ESA was used in 1L therapy among 83.3% pts in US/CAN and 87.7% in UK/EU. Image:Summary/Conclusion: ESA-based regimens were the most common treatment for anemia management in pts with LR-MDS; >1/3 of pts were TD before initiating 1L ESA therapy. A considerable proportion of 1L ESA-treated pts reinitiated ESA in 2L despite the majority having discontinued 1L treatment due to planned therapy completion, pt decision, or progressive disease. This real-world study suggests that there is an unmet need in addressing anemia burden with ESAs, and novel and effective treatments are needed.

S146: VENETOCLAX IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P DELETION: 6-YEAR FOLLOW-UP AND GENOMIC ANALYSES IN A PIVOTAL PHASE 2 TRIAL

Background: Patients (pts) with chronic lymphocytic leukemia (CLL) with del(17p) and/or mutated TP53 have adverse prognosis and are a population of interest for improving outcomes. In a pivotal Phase 2 study (NCT01889186) evaluating venetoclax (Ven; a selective oral BCL2 inhibitor) monotherapy in del(17p) CLL (N=158), overall response rate (ORR) was 77% (median time on study, 26.6 mo; Stilgenbauer. J Clin Oncol. 2018;17:1973). Aims: To report final analyses after 5 y from last pt enrolled, including post-hoc subgroup analyses of prognostic markers. Methods: Adults with R/R or previously untreated del(17p) CLL received Ven 400 mg (via ramp-up) orally daily until progressive disease (PD) or intolerance. Main cohort comprised pts with R/R CLL; safety expansion cohort included pts with R/R or untreated CLL. Primary endpoints were ORR (main) and safety (safety expansion). Peripheral blood (PB) and bone marrow (BM) were analyzed pre-Ven for somatic mutations via targeted next-generation sequencing (NGS). Minimal residual disease (MRD) was assessed by flow cytometry, ASO-PCR, and/or clonoSEQ NGS; undetectable MRD (uMRD) is reported at <10−4. Results: In all, 158 pts received Ven (main, n=107; safety expansion, n=51). Data cutoff was 15 December 2020. Pts had median 2 (range, 0−10) prior lines of therapy (LOT); 5 had untreated CLL (first-line [1L]). Common mutations (n/N) were TP53 (113/138 [82%]; 35/113 [31%] had >1), SF3B1 (28/137 [20%]) NOTCH1 (22/137 [16%]), ATM (11/120 [9%]), and BIRC3 (6/120 [5%]); 93/115 (81%) had unmutated IGHV and 118/158 (75%) had ≥20% del(17p). Median time on Ven was 27.4 (range, 0–79.3) mo. At study close, 77 pts were alive; 26 remained on Ven. No new safety signals were observed. With 70 mo median follow-up (f/u), investigator-assessed ORR was 77% (95% CI, 70−84); 21% achieved complete remission (CR)/CR with incomplete blood count recovery (CRi). Median duration of response was 39.3 mo (95% CI, 31.1−50.5); 28% had ongoing response at 60 mo. Of 61 evaluable pts with a PB MRD assessment, 25% had uMRD at ~2 y (24−30 mo). Median progression-free survival (mPFS) was 28.2 mo (95% CI, 23.4−37.6; Figure). Median overall survival (mOS) was 62.5 mo (95% CI, 51.7−NR; 5-y PFS and OS rates, 24% and 52%). Of 5 1L pts, 4 were alive and progression-free. In pts with CR/CRi (n=37), mPFS was 62.2 mo (95% CI, 53.1−NR); in pts with partial remission (PR)/nodular PR (n=85), mPFS was 27.6 mo (95% CI, 22.8−38.0). Overall, 98/158 (62%) pts had PD, including 24/158 (15%) with Richter transformation. Of pts with PD (n=98) or whose disease was refractory to Ven (n=11), 73 received another LOT, most commonly ibrutinib (n=41); mOS from ibrutinib initiation was 28.0 mo. In pts with both del(17p) and TP53 mutation (n=111) vs those with either (n=19), ORR was 75% vs 79%; mPFS was 27.4 vs 22.8 mo (P=.8). In pts with mutated (n=22) vs unmutated IGHV (n=93), mPFS was 40.4 vs 26.9 mo (P=.11). No significant difference in ORR, PFS, or OS was observed in pts with vs without ≥20% del(17p), >1 TP53 mutation, NOTCH1, ATM, or BIRC3 mutations; mPFS was shorter in pts with mutated SF3B1 (n=28) vs without (n=109; 16.4 vs 30.2 mo [P=.0071]). Multivariate analysis is ongoing. Image:Summary/Conclusion: At end of study (median f/u, 70 mo), 48% of pts were alive, 24% were progression-free, and 16% remained on Ven, confirming the long-term activity of Ven in this high-risk population with del(17p) CLL and median 2 prior LOT. Except SF3B1 mutation, other adverse features (eg, >1 TP53 mutation, NOTCH1 mutations, unmutated IGHV) did not influence outcomes with Ven treatment in this cohort.

P621: SUBCLONAL EVOLUTIONARY TRAJECTORIES IN IGLV3-21R110 CHRONIC LYMPHOCYTIC LEUKEMIA

Background: There is a large body of evidence supporting the importance of antigenic interactions of the B cell receptor (BCR) with self or non-self antigens that make this receptor and its signaling machinery one of the most important drivers in many types of non-Hodgkin lymphoma. This has led to a new pathobiological understanding of these diseases as well as novel therapeutic strategies. Chronic lymphocytic leukemia (CLL) is a prototype disease in which BCR-BCR self-interactions induce autonomous signaling. While virtually all CLL BCR are prone to autonomous signaling induced by such contacts, the IGLV3-21R110 molecular interaction found in 10% of CLL patients defines a distinct clinical subset of patients that show a very aggressive clinical course despite novel treatments. In these cases, a single amino acid replacement from G to R located at the boundary of variable and constant region of the BCR is an important tumor promotor driving neoplastic B cell growth. Aims: It has been unclear how the R110 point mutation is generated. We set out to investigate the trajectory of the malignant CLL B cell clone and subclonal heterogeneity in IGLV3-21R110 cases in order to gain insight into the molecular pathogenesis of this unique amino acid exchange. Methods: We screened an initial CLL cohort of 127 cases by flow cytometry using an anti-IGLV3-21R110 antibody. Positive cases were subjected to immunosequencing of the light chain locus via amplicon based next-generation sequencing and characterized for typical chromosomal deletions and driver gene mutations. Results: We identified twelve CLL cases with IGLV3-21R110. Of these, we found a higher frequency of mutations in ATM (42%) and SF3B1 (25%) than in unselected CLL cohorts, which is in line with findings reported by others. As expected, the repertoire of healthy B cells in the blood of IGLV3-21R110 CLL patients contained IGLV3-21 light chains with the wild-type G110 residue, indicating that the R110 exchange is of acquired nature. Interestingly, in half of the IGLV3-21R110 CLL cases we found additional clones with IGLV3-21 and R110, including cases with divergent J gene cassettes originating from different pre-B cells. This indicates that the R110 exchange may occur multiple times within independent B cell clones in the same patient. Due to the unique location at the variable-constant boundary and the high correlation of the IGLV3-21R110 patient subset with mutations of genes involved in DNA repair (ATM/SF3B1), R110 is likely generated as a result of incorrect V-J recombination facilitated by the error-prone-environment. Summary/Conclusion: Our work indicates that the G110R point mutation results from incorrect V-J rearrangements in the error-prone environment that may be induced by SF3B1 and ATM mutations associated with this CLL subset. We found evidence for a branching light chain evolution that leads to a striking convergence of separate clones on the identical light chain amino acid exchange in the same patient. This strongly emphasizes the role of this mutation as a tumor promotor involved in malignant transformation of this subset of CLL.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia

AbstractDetailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3′ end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3′ splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).