ALBERT

Description: Introduction: Idelalisib (IDELA) is a first-in-class PI3Kδ inhibitor that is approved for use in combination with rituximab for patients with relapsed or refractory chronic lymphoid leukemia (R/R CLL) in the United States and in combination with rituximab or ofatumumab for the same indication in the European Union. Clinical trials evaluating IDELA in the first line setting for CLL were prematurely terminated due to an increased incidence of serious adverse events (AEs) and mortality; therefore, IDELA is not indicated as a first line therapy for CLL. The safety signal observed in the treatment naïve setting has produced a prevailing view that early line use of IDELA may also lead to inferior clinical outcomes. To address this question, we evaluated the clinical benefit:risk balance for IDELA in early vs. later lines of use in the relapsed setting. Objective: To investigate clinical outcomes across sub-populations of IDELA + anti-CD20-treated patients with R/R CLL based on number of prior chemo-immunotherapy treatment regimens. Methods: Using clinical outcomes data collected in Gilead-sponsored clinical trials of patients with R/R CLL treated with IDELA + anti-CD20 (312-0116/0117, N=110, Furman et al., N. Engl. J. Med. 2014; 370:997-1007, and 312-0119, N=173, Jones et al., Lancet Haematol. 2017; 4:e114-e126), we retrospectively compared the overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and AE profile for patients previously treated with 1 prior regimen (IDELA given in the second line setting, 2L), 2 prior regimens (3L), or ≥3 prior regimens (≥4L). PFS and OS were estimated using the Kaplan-Meier method and groups were compared using a log-rank test. The incidence of AEs was compared using the Kruskal Wallis test. Results: Among 283 patients treated with IDELA + anti-CD20, 49 (17.3%) received IDELA 2L, 69 (24.4%) received it 3L, and 165 (58.3%) received it ≥4L. Patient characteristics were similar except that patients in the ≥4L group presented with Rai stage III/IV disease more frequently compared to the 2L and 3L groups (70.3% vs. 61.3% and 57.9%, respectively, Table 1). ORR was similar irrespective of the number of prior regimens (85.7% for the 2L group, 73.9% for the 3L group, and 80% for the ≥4L group, p=0.2866); however, PFS and OS were longer in the 2L group compared to the 3L and ≥4L groups (median PFS= 31.5 months, 16.6 months, and 17.3 months, respectively, [Figure 1] and median OS=47.4 months, not reached (NR), and 34.6 months, respectively). No statistically significant difference was observed across treatment setting groups in the incidence of grade 3/4 key treatment-emergent AEs, including cough, diarrhea, infection, transaminitis, and colitis (Figure 2). Conclusion: These analyses indicate that patients with R/R CLL experience comparable or improved efficacy and have a similar safety profile when IDELA + anti-CD20 regimens are used 2nd line as compared to later line, after chemo-immunotherapeutic regimens. The longer PFS and OS times for patients treated with IDELA in the 2L may reflect shorter time since diagnosis, lower stage disease, or less disease resistance in this group, but also suggest that the efficacy benefit of IDELA + anti-CD20 is greater and may therefore better offset the potential toxicity in this patient sub-group. These findings support the use of IDELA + anti-CD20 in the 2L+ setting for patients with R/R CLL following chemo-immunotherapy. Disclosures Brown: Abbvie: Consultancy; TG Therapeutics: Consultancy; Sunesis: Consultancy; Gilead: Consultancy, Research Funding; Genentech: Consultancy; Boehringer: Consultancy; Pharmacyclics: Consultancy; Verastem: Consultancy, Research Funding; Loxo: Consultancy; Sun Pharmaceutical Industries: Research Funding; Invectys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy. Ye:Gilead Sciences, Inc.: Employment, Equity Ownership. Xing:Gilead Sciences, Inc.: Employment. Roudet:Gilead Sciences, Inc.: Employment. Ruzicka:Gilead Sciences, Inc.: Employment. O'Brien:TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy; Alexion: Consultancy; Acerta: Research Funding; Astellas: Consultancy; GlaxoSmithKline: Consultancy; Aptose Biosciences Inc.: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding; Regeneron: Research Funding; Pfizer: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy; Kite Pharma: Research Funding; Amgen: Consultancy; Vaniam Group LLC: Consultancy.

Description: Introduction: Idelalisib (IDELA, Zydelig®) is the first-in-class PI3Kδ inhibitor and is approved in the U.S. as an oral monotherapy for relapsed / refractory follicular lymphoma (R/R FL) after at least two prior lines of systemic therapy. IDELA's regulatory approval was based on a phase 2, open-label clinical trial in 125 patients with R/R indolent non-Hodgkin's lymphoma (Gopal et al., NEJM, 2014) and outcomes in the FL subgroup were published by Salles et al. (Haematologica, 2017). The current study evaluates the characteristics and treatment patterns of patients treated with IDELA for R/R FL in a real-world setting. Methods: Adult patients diagnosed with R/R FL (grades 1, 2, and 3a) and treated with IDELA within the US Oncology Network (USON) between 7/1/2014 to 6/30/2018 were analyzed retrospectively. Patient data were obtained from USON's structured electronic health records' system, iKnowMed (iKM)TM. Manual chart review (ChR) was used to determine physician response and to confirm IDELA treatment patterns. Overall survival (OS) and progression-free survival (PFS) were estimated using Kaplan-Meier methods. Descriptive statistics were generated for outcomes of interest, including duration of therapy (DoT), median follow-up, and adverse event (AE) frequency. Results: A total of 124 patients with FL and prescribed IDELA were identified in iKM TM. After Chr confirming the diagnosis of follicular lymphoma diagnosis and initiation of IDELA, 88 patients were retained for analysis. Median age of patients was 68.9 years, with 52.3% female and the majority white and non-Hispanic (90.9% and 93.2%, respectively, Table 1). The most common regimens immediately prior to IDELA initiation were bendamustine + rituximab (22.7%), rituximab (17%), and rituximab maintenance (11.4%). Eighty-six (97.7%) patients had co-morbidities categorized as vascular (50%), endocrine (33%), respiratory (13.6%), or cardiac (12.5%). Thirteen (14.8%), 21 (23.9%), and 54 (61.4%) patients initiated IDELA in second line (2L), 3L, and 〉4L, respectively. Baseline lab values at IDELA initiation were similar regardless of line of therapy (LOT). mDOT was 5.5 mos. for the entire population and was similar across all LOTs (4.1 mos., 6.1 mos., and 5.5 mos. in 2L, 3L, and 〉4L, respectively). AEs were noted in 45.5% with the most common being gastrointestinal (31.8%) and dermatologic (10.2%). Respiratory and infectious AEs were noted in 2.3% and 1.1%, respectively, although Pneumocystis jirovecii pneumonia (PJP) prophylaxis was rarely prescribed (2.3%). Toxicity as a reason for IDELA discontinuation varied in frequency across LOT and was more common in 2L compared to 3L and 〉4L (91.7% compared to 43.8% and 46.9%, respectively). With a median follow-up of 18.6 months for the population, the mPFS was 11.4 mos. [95%CI: 8.5,17.0] and mOS was 32.5 mos. [95% CI: 25.3,NR]. Stratified by LOT, median follow-up time, mOS, and mPFS were greater in 2L (30.8 mos., NR [95% CI: 27.37,NR], and 29.0 mos. [95% CI: 8.6,NR], respectively) than in 3L or 〉4L (3L: 17.9 mos., 29.4 mos. [95%CI: 18.6,NR], and 17.5 mos. [95% CI: 6.1,NR]; 〉4L: 16.5 mos., 25.3 mos. [95%CI: 13.5.,NR], and 8.6 mos. [95% CI: 6.1,12.6], respectively, Figures 1 and 2). Conclusion: Findings from this analysis suggest that R/R FL patients treated with IDELA in a real-world setting experience a similar mDOT and mPFS as those treated in the clinical trial setting (Salles et al., Haematologica, 2017). Patients treated in 2L demonstrated longer PFS and OS compared to later lines, but also experienced increased IDELA discontinuation due to toxicity, perhaps reflecting a lower incidence of progressive disease in earlier treatment lines, or a more immunocompetent population leading to higher rates of autoimmune AEs. Use of PJP prophylaxis in IDELA-treated patients was uncommon, an observation suggesting an opportunity for provider education. Our findings enhance available data on relapsed FL patient outcomes in real-world clinical practice and support the use of IDELA in patients with R/R FL after at least 2 systemic therapies. Disclosures Andorsky: Gilead: Research Funding; Genetech: Research Funding; CTI: Research Funding; AstraZeneca: Consultancy; Celgene: Research Funding. Chan:Gilead Sciences, Inc.: Employment, Equity Ownership. Clark:McKesson: Consultancy, Employment, Equity Ownership. Ruzicka:Gilead Sciences, Inc.: Employment. Robert:McKesson: Employment. Awan:Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy; Genentech: Consultancy; Sunesis: Consultancy; Gilead: Consultancy. OffLabel Disclosure: Idelalisib is a PI3 kinase inhibitor indicated for the treatment of patients with relapsed follicular B-cell lymphoma who have received at least two prior systemic therapies. Some patients in this observational study used Idelalisib after one prior systemic treatment.

Description: Juvenile Myelomonocytic Leukemia is the most common pediatric myeloproliferative neoplasm (MPN). JMML is characterized by myeloid populations with mutually-exclusive mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. JMML is notable among pediatric MPNs as being refractory to chemotherapy and having a 50% relapse rate following allogeneic hematopoietic stem cell (HSC) transplantation. As such, there is an urgent need for novel JMML therapies. The recent discovery of yolk sac myeloid lineages that persist into adulthood independently of bone marrow HSC contributions suggests a mechanism for JMML relapse following HSC transplantation. In this study, we sought to determine whether yolk sac HSC-independent myeloid progenitors bear hallmarks of MPN in a mouse model of JMML. Using the Vav1 promoter-directed Cre recombinase, we generated a mouse model of JMML that expresses the PTPN11D61Y gain of function mutation in all waves of embryonic and adult hematopoiesis, including yolk sac myeloid progenitors that emerge prior to and independently from HSCs. PTPN11D61Y/+; VavCre+ mice are viable, born at expected Mendelian ratios, and develop peripheral blood monocytosis as early as 4 weeks of age. Given this early onset, we hypothesized MPN may develop in these mice during embryonic development. E14.5 fetal liver progenitors from PTPN11D61Y/+; VavCre+ embryos displayed marked GM-CSF hypersensitivity in methylcellulose colony forming assays (Figure-1A), possessed hyperactive Ras-Erk pathway signaling (Figure-2), and had a skewed progenitor distribution with a greater proportion of megakaryocyte-erythroid progenitors (63.5% vs. 50.1%, p

Description: Key Points Compared with ubiquitously expressed PI3K p110α, genetic inhibition of PI3K p110δ uniquely normalizes mutant Shp2-induced GM-CSF hypersensitivity. Potent pharmacologic inhibitors of PI3K p110δ cooperate with MEK inhibition to reduce mutant Shp2-induced hyperproliferation.

Description: The childhood leukemia, juvenile myelomonocytic leukemia (JMML), is a lethal disease of young children characterized by spontaneous growth of peripheral blood hematopoietic progenitors and hypersensitivity of hematopoietic progenitors to the cytokine GM-CSF. Notably, hematopoietic progenitors from JMML patients do not typically demonstrate hypersensitivity to the cytokine IL-3. Mutations in the RAS and NF1 genes have long been recognized as pathogenic in this disease, and more recently, mutations in the PTPN11 gene, which encodes Shp-2, a non-receptor protein tyrosine phosphatase, have also been found commonly in leukemic cells from children with JMML. We hypothesized that the mutant Shp-2 molecules observed in JMML patients would induce hypersensitivity of hematopoietic progenitors to GM-CSF. To examine this hypothesis, we subcloned the WT Shp-2 cDNA and three mutant Shp-2 cDNAs found in leukemic cells from children with JMML (E76K, D61V, and D61Y), into the murine stem cell retroviral plasmid pMIEG3 in tandem with EGFP. Each vector was sequenced to verify the desired point mutation and to rule-out unwanted mutations. Murine bone marrow low density mononuclear cells were subjected to fibronectin-assisted retroviral transduction and sorted for EGFP positive cells using fluorescence activated cell sorting. EGFP positive cells were plated into progenitor assays with increasing concentrations of GM-CSF (0, 0.01 0.1, 1, and 10 ng/mL). As predicted, transduction with each of the Shp-2 mutations induced hypersensitivity to GM-CSF compared to vector alone or to WT Shp-2, as evidenced by significantly higher % maximal colony formation at each GM-CSF dose tested (Figure 1). We next conducted progenitor assays using EGFP positive cells with increasing concentrations of IL-3 (0. 0.01, 0.1, 1, and 10 U/mL). Although hematopoietic progenitors from JMML patients have not been described to be hypersensitive to IL-3, surprisingly, we have observed in preliminary studies that introduction of each of the Shp-2 mutations induced hypersensitivity to IL-3 in a similar fashion to that observed with GM-CSF (Figure 2). These data demonstrate that somatic PTPN11 mutations observed in children with JMML induce hematopoietic cell growth aberrancies in a manner overlapping with the classical description of JMML, yet also induce unique hematopoietic cell growth characteristics based on the observed hypersensitivity to IL-3. These findings suggest that signaling pathways in addition to the Ras-MAPK cascade may be dysregulated in PTPN11 mutation-bearing hematopoietic cells and provide a novel model for the investigation of new therapeutics in JMML. Current studies are ongoing to examine the effect of these mutations on in vivo hematopoiesis and leukemogenesis.

Description: Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1+Mac1+ myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3– or BCR homology domain–deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP−/− and p85α−/− mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

Description: Neurofibromatosis type I (NF1) is a congenital disorder resulting from loss-of-function of the tumor suppressor gene, NF1. 50% of NF1 patients have osseous manifestations, including short stature, scoliosis, and reduced bone mineral density. Osteoclasts are hematopoietic stem cell-derived cells that function to resorb bone. We recently reported that osteoclasts derived from NF1 patients and Nf1 heterozygous (Nf1+/−) mice have elevated migration, adhesion, and bone resorption and our studies indicate that the gain-of-function of the Nf1+/− osteoclasts is, at least in part, caused by hyperactivation of macrophage colony-stimulating factor (M-CSF)-stimulated Ras, phosphoinositol-3-kinase (PI3K), and Erk. Rho GTPases function downstream of Ras and PI3K and act as binary switches, cycling between an inactive (GDP-bound) and active (GTP-bound) state, to regulate osteoclast actin ring formation, bone resorption, and development of filamentous actin structures associated with migration and adhesion. We hypothesized that hyperactivation of Rac1, Rac2, or both Rac1 and Rac2 contribute to the increased osteoclast function observed in Nf1+/− mice and NF1 patients. To examine this hypothesis, we intercrossed Nf1+/− mice with conditional Rac1flox/floxMxcre+ mice or with Rac2−/− mice to generate WT, Nf1+/−, Rac1−/−, Nf1+/−;Rac1−/−, Rac2−/−, and Nf1+/−;Rac2−/− mice. Genetic disruption of Rac1, but not of Rac2, restored the increased colony forming unit-macrophage (CFU-M), tatrate resistant acid phosphate+ (TRAP+) CFU-M, osteoclast migration, and bone resorption observed in Nf1+/− cultures. Osteoclast bone resorbing capacity is dependent on the organization of the actin cytoskeleton into a large f-actin-rich structure referred to as the sealing zone. The podosome belt evolves into the sealing zone in actively resorbing osteoclasts. A significantly higher level of belt formation, seen in mature osteoclasts, was observed in Nf1+/− cultures as compared to WT. Upon genetic deletion of Rac1, the Nf1+/−;Rac1−/− osteoclasts demonstrated belt formation at a similar level to that of WT osteoclasts. These data indicate that Rac1 plays an essential role in functional f-actin organization and suggest that inhibition of Rac1 in the setting of Nf1 haploinsufficiency is able to normalize osteoclast hyperactivity by correcting the cytoskeletal organization of f-actin-based structures. Mechanistically, Rac1 deficiency normalized M-CSF-stimulated phospho-Erk and phospho-Akt and pharmacologic inhibition of MEK and PI3K using PD98059 or Ly294002, respectively, normalized Nf1+/− osteoclast development and maturation. The critical role of Rac1, but not of Rac2, in osteoclast function is significant as it suggests that the Rac GTPases contribute non-redundant functions in various myeloid cell types and imply that blocking Rac1 function, while sparing that of Rac2, may provide a level of specificity to therapeutics for skeletal diseases. Collectively, these data demonstrate that Rac1 critically contributes to increased osteoclast function induced by haploinsufficiency of Nf1 and imply that Rac1 may be a rational therapeutic target for dysplastic and erosive bone diseases.

Description: Abstract 2420 Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in individuals with acute myeloid leukemia (AML). Based on the finding that the protein tyrosine phosphatase, Shp2, interacts with WT FLT3 tyrosine (Y) 599, which is commonly duplicated in FLT3-ITDs, we hypothesized that increased recruitment of Shp2 to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and aberrant STAT5 activation. Co-immunoprecipitation studies demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Additionally, we found that genetic disruption of Ptpn11, the gene encoding Shp2, significantly reduced N51-FLT3-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation in vitro. To investigate these findings further, Lin- bone marrow cells from Shp2flox/flox;Mx1Cre+ animals were retrovirally transduced with N51-FLT3, sorted to homogeneity, and transplanted into lethally irradiated congenic recipients. Transplanted animals were treated with polyI:polyC to delete Shp2 or with phosphate buffered saline (PBS control) 4 – 6 weeks following transplantation, and animals were followed temporally. The majority of PBS-treated animals (16/18) died of hematologic malignancy. In contrast, animals with Shp2 deletion (polyI:polyC-treated, n=16) succumbed to malignant disease less frequently (10/16), demonstrated a significantly prolonged survival (p=0.024 by log-rank test), and had smaller spleen sizes compared to the PBS-treated animals. Notably, Y599 has been shown to recruit Shp2 to WT FLT3 and mutation of Y599 to phenylalanine (F) within WT FLT3 causes a reduction in FL-stimulated cell proliferation. Thus, we generated point mutants including N51-Y599F1 bearing the Y to F mutation at the first Y599 and N51-Y599F1/2 bearing Y to F mutation at both the first and duplicated Y599. Murine bone marrow low density mononuclear cells were transduced with each construct and subjected to 3H-thymidine incorporation and immunoblot for proliferation and STAT5 activation, respectively. While mutation of the first Y599 alone failed to reduce proliferation or STAT5 phosphorylation, mutation of both the first and duplicated Y599 significantly reduced cellular proliferation and phospho-STAT5 levels. To investigate molecular mechanisms underlying how constitutive association of Shp2 with STAT5 may promote FLT3-ITD-induced leukemogenesis, we utilized the human FLT3-ITD positive AML-derived cell line, MV411. While previous studies have demonstrated nuclear localization of Shp2 in AML samples, the role of nuclear Shp2 in leukemia has never been investigated. We utilized in situ immunofluorescence to examine nuclear distribution of Shp2 and potential co-localization with phospho-STAT5. Strong nuclear expression of Shp2 was observed in MV411 cells, and upon merging of images, nuclear Shp2 co-localized strongly with nuclear phospho-STAT5, suggesting that Shp2 may work with STAT5 within the nucleus to enhance gene expression promoting leukemogenesis. We chose to examine the BCL2L1 promoter, a STAT5-responsive promoter which regulates expression of the prosurvival protein, Bcl-XL. Using chromatin immunoprecipitation assays, we found Shp2 is present at functional interferon-g activation sites (GAS) within the BCL2L1 promoter. Furthermore, knockdown of Shp2 in MV411 cells resulted in reduced phospho-STAT5 levels and reduced BCL2L1 promoter-directed luciferase expression. Moreover, using a novel small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was significantly reduced in a dose-dependent manner. Our findings suggest that constitutive association of Shp2 with N51-FLT3 promotes hyperproliferation and that either genetic disruption of Shp2 expression or mutation of the Shp2 binding sites on N51-FLT3 significantly abrogates N51-FLT3-induced hyperproliferation, STAT5 hyperactivation, and N51-FLT3-induced hematologic malignancy in vivo. Furthermore, Shp2 and STAT5 appear to work functionally in the nucleus to promote STAT5-responsive, pro-leukemogenic gene expression. Collectively, these studies demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML. Disclosures: No relevant conflicts of interest to declare.