Gurbuxani, Sandeep K.

Introduction: TP53 mutations are found in 18% of adult patients (pts) with acute lymphoblastic leukemias (ALL), and portends poor prognosis (Saygin, Blood Cancer Discov 2024). TP53 mutations are often associated with low hypodiploidy and Li Fraumeni syndrome (germline) in children. However, we and others have shown that TP53-mutant (TP53-m) ALL arises from pre-existing TP53-m clonal hematopoiesis in adults as opposed to germline origin. The impact of co-occurring mutations, TP53 allelic frequency (AF) and different types of treatment on outcomes of TP53-m ALL are not fully characterized. We set out to investigate the clinical and molecular predictors of outcomes in adults with TP53-mutant ALL. Methods: We retrospectively studied 107 patients with TP53-m ALL treated at seven different academic centers in North America. Genetic profiling of pre-treatment bone marrow samples was performed with a comprehensive panel covering 147 myeloid and lymphoid genes. We also performed RNA-sequencing to identify fusions that define ALL subtypes. Single nucleotide polymorphism (SNP) array was used to define low hypodiploidy. Results: The median age at diagnosis was 59 years (range, 10-80), 42% were female, 70% White. 89% of pts had B-ALL while 11% had T-lineage ALL. 24% of pts were classified to have therapy-related ALL based on their history of exposure to genotoxic therapy for another cancer. 45% of pts had hypodiploid karyotype, 79% of whom were low-hypodiploid. 7% had hyperdiploid karyotype and 5% had BCR::ABL1. 11 pts had two different TP53 variants. 90% of TP53 variants involved the DNA binding domain. Most frequently observed co-occurring mutations involved CDKN2A (20%), NOTCH1 (12%) and RB1 (10%) genes. 71% of pts were treated with standard cytotoxic chemotherapy, while the remaining pts received newer antibody-based therapies (i.e., inotuzumab, blinatumomab) or venetoclax in first-line setting. 11% of pts received allogeneic hematopoietic cell transplant (HCT) in first complete remission (CR1) while 3% received HCT after relapse (CR2 or CR3). When patients with vs without low hypodiploidy were compared, we did not observe a significant difference in overall survival (OS) as outcomes were poor in both groups (median, 17 vs 19 months, p= 0.89). However, pts who had TP53 AF >0.5 had significantly worse OS when compared to pts with TP53 VAF <0.5 (13 vs 25 months, p= 0.03). Conclusions: TP53-m ALL is a very high-risk disease subtype and its dismal outcome is independent from presence of co-existing low hypodiploidy. Loss of heterozygosity, characterized by TP53 AF >0.5 is associated with even worse survival. New therapies are urgently needed to improve outcomes in this subset.

Abstract The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1α, and downregulation of LKB1 and increased levels of HIF1α were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. Significance: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1a and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression. This article is highlighted in the In This Issue feature, p. 1307

Nearly 20% of patients with myelofibrosis progress to blast phase disease; an aggressive form of acute myeloid leukemia. Although previous studies have implicated loss of TP53 or JARID2 in progression, by and large the genetic events that lead to conversion to blast phase remain unknown. To identify genes whose loss drives progression, we performed a focused CRISPR/Cas9 screen in which murine Jak2V617F bone marrow cells expressing Cas9 were transduced with two separate sgRNA libraries of known tumor suppressor genes and subjected to colony replating assays. Transduction of one of the two libraries led to serial replating and enhanced self-renewal of the Jak2V617F cells. Subsequent DNA sequencing revealed enrichment of all four guides targeting STK11, the gene that encodes LKB1 which regulates a number of key cellular pathways including energy utilization by activation of AMPK. To confirm that loss of Stk11 is the event that leads to increased clonogenicity, we collected cells from Jak2V617F/Vav-Cre+ and control Vav-Cre+ mice and induced Stk11 knockout by electroporating Cas9-Stk11 sgRNA ribonucleoprotein complexes. Consistent with the screening results, only Jak2V617F Vav-Cre+ cells with Cas9-Stk11 sgRNA showed serial replating. To determine whether Stk11 is required for growth of cells with a different driver of enhanced JAK/STAT signaling, we doubly transduced Stk11 homozygous floxed bone marrow cells with MPLW515L-mCherry and Cre-GFP to delete Stk11. As expected, cells with both MPLW515L and Cre recombinase showed enhanced self-renewal, while singly infected control cells failed to replate. These results demonstrate that activation of JAK/STAT signaling can overcome the requirement for Stk11 in normal hematopoiesis and suggest that STK11 loss may be a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling. We next investigated the mechanism by which loss of STK11 cooperates with enhanced JAK/STAT signaling to promote leukemia. RNA-sequencing of wild-type, Stk11+/+/ MPLW515L, and Stk11-/-/MPLW515L hematopoietic cells revealed enrichment of a number of pathways related to hypoxia, oxidative phosphorylation and mitochondrial translation in cells lacking LKB1. Western blot assays confirmed activation of mTOR signaling as well as HIF1a stabilization and pathway activation, both of which have been reported to lie downstream of LKB1 loss. We also performed a number of studies to determine the relevance of reduced LKB1 expression to leukemic progression. First, we induced deletion of Stk11 in mice that were transplanted with HSPCs expressing MPLW515L after development of the MPN phenotype. Loss of Stk11 caused a rapid lethality that was associated with enhanced bone marrow fibrosis and osteosclerosis. We also observed accumulation of leukemic blasts in small clusters consistent with AML transformation arising in the spent phase MPN. Additionally, we deleted STK11 by CRISPR/Cas9 in primary MPN patient samples and monitored their engraftment in immunocompromised mice. We observed enhanced engraftment and increased reticulin fibrosis and osteosclerosis in mice that received the STK11 edited cells compared to those with non-targeted sgRNA. Third, we compared the expression of STK11 in paired blast and chronic phase myelofibrosis patient samples by RT-PCR. Consistent with the hypothesis that loss of STK11 facilitates leukemia, we found that its expression was decreased by more than 50% in five of seven paired post-MPN AML patient samples, with two having STK11 levels below 20%. We further validated downregulation of LKB1 by immunohistochemistry on paired chronic and blast phase MPN specimens and observed little staining in the blast phase specimens. Finally, to further show that the mechanism of in vitro enhanced self-renewal is related to leukemia progression, we stained the paired marrows for HIF1a and saw a dramatic increase in staining at the AML phase. We also analyzed RNA-seq data of paired chronic versus blast phase MPNs specimens and observed that there is a strong congruence of enriched pathways that are associated with the in vitro mouse HSPC phenotype and the human blast phase progression, such as oxidative phosphorylation and hypoxia. Together, our study demonstrates that loss of LKB1/STK11 promotes transformation of cells with activated JAK/STAT signaling and that STK11 is a prominent candidate tumor suppressor gene in post-MPN AML. Disclosures Gurbuxani: UpToDate: Honoraria. Hoffman:Dompe: Research Funding; Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees. Levine:Astellas: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Rampal:Galecto: Consultancy; Incyte: Consultancy, Research Funding; Constellation: Research Funding; Stemline: Consultancy, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; CTI Biopharma: Consultancy; Abbvie: Consultancy; Pharmaessentia: Consultancy; Promedior: Consultancy; Blueprint: Consultancy.

Abstract Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Abstract We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.

Abstract Purpose: Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis. Patients and Methods: Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis. Results: In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients. Conclusions: Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis. See related commentary by Piszczatowski and Steidl, p. 4868