Abstract: Background: MRD status is an established predictive marker for progression-free survival (PFS) in CLL following chemoimmunotherapy as well as for fixed-duration treatment with venetoclax and an anti-CD20 antibody. To date, this relationship has not been explored for the combination of Ibr+Ven, an all-oral, once-daily, fixed-duration treatment with complementary mechanisms of action that work synergistically to eliminate CLL subpopulations in distinct tumor compartments. In the primary analysis of the phase 3 international GLOW trial, independent review committee (IRC)-assessed PFS for Ibr+Ven was superior to Clb+O (hazard ratio, 0.216; p & lt; 0.0001). Herein we further investigate MRD outcomes at the time of the GLOW primary analysis. Methods: GLOW (NCT03462719) enrolled patients (pts) aged ≥65 years or 18-64 years with cumulative illness rating scale score & gt;6 or creatinine clearance & lt;70 mL/min. Pts with del(17p) or known TP53 mutations were excluded. Pts were randomized 1:1, stratified by IGHV mutational and del(11q) status, to Ibr+Ven (3 cycles of ibrutinib lead-in, followed by 12 cycles of Ibr+Ven) or 6 cycles of Clb+O. The primary end point was PFS assessed by IRC and rate of undetectable MRD (uMRD; & lt;10 -4) was a secondary end point; additional MRD analyses reported here are exploratory. MRD samples were collected for responders every 3-4 months in peripheral blood (PB) and at Months 9 and 18 in bone marrow (BM). MRD was evaluated using next-generation sequencing (NGS; clonoSEQ) and 8-color flow cytometry. PB/BM concordance was calculated for pts with uMRD in PB at end of treatment plus 3 months (EOT+3) who had a paired BM sample. Analysis of PFS by MRD status includes pts with known MRD status at EOT+3 and no prior progression, death, or withdrawal. Results: 106 pts were randomized to Ibr+Ven and 105 to Clb+O. Median age was 71.0 years, 51.7% had confirmed unmutated (u)IGHV, 18.0% had del(11q), and 4.3% had a TP53 mutation. Median follow-up was 27.7 (range, 1.7-33.8) months. MRD results are all via NGS and reported for EOT+3 unless otherwise noted. MRD Results at 10 -4: Rate of uMRD was significantly higher for Ibr+Ven vs Clb+O in BM (51.9% vs 17.1%; p & lt; 0.0001) (Fig A) and in PB (54.7% vs 39.0%; p = 0.0259). PB/BM uMRD concordance with Ibr+Ven was 92.9%. In the Ibr+Ven arm, 65.9% (27/41) of pts with a complete response (CR) or CR with incomplete marrow recovery (CRi) and 54.9% (28/51) with a partial response achieved uMRD in BM; rates for Clb+O were 33.3% (4/12) and 16.9% (13/77), respectively. BM uMRD rates were higher for Ibr+Ven vs Clb+O across prespecified subgroups, including bulky disease (≥5 cm), del(11q), and uIGHV . In the Ibr+Ven arm, BM uMRD was higher for uIGHV (58.2%) vs mutated IGHV (44.4%). With Ibr+Ven, 84.5% (49/58) of pts maintained PB uMRD from EOT+3 to EOT+12 vs 29.3% (12/41) with Clb+O. For pts with detectable MRD after Ibr+Ven (n = 30), MRD levels remained stable for most patients from EOT+3 to EOT+12 (Fig B). MRD Results at 10 -5: Rate of uMRD & lt;10 -5 was higher for Ibr+Ven vs Clb+O in BM (40.6% vs 7.6%) (Fig A), including pts with uIGHV (45.5% vs 5.6%). With Ibr+Ven, PB/BM uMRD concordance at & lt;10 -5 was 90.9% (40/44). Among pts with uMRD & lt;10 -4 in the Ibr+Ven arm, the majority achieved & lt;10 -5 in PB (79.3%) and BM (78.2%), including pts with uIGHV. uMRD & lt;10 -5 in PB was largely sustained from EOT+3 to EOT+12 with Ibr+Ven (80.4% [37/46] of pts) but not Clb+O (26.3% [5/19] of pts) (Fig B). PFS by MRD Status at 10 -4 : In the Ibr+Ven arm, PFS rate during the first 12 months after EOT was & gt;90% for pts with uMRD as well as pts with detectable MRD. In contrast, pts in the Clb+O arm with detectable MRD in PB relapsed more quickly than those with uMRD (Fig C). PFS trends were similar according to MRD status in BM (Fig D). Note that not all pts in the Ibr+Ven arm had 12 months' follow-up post-EOT. Conclusion: All-oral, once-daily, fixed-duration Ibr+Ven demonstrated superior uMRD responses that were deeper and better sustained post-treatment vs Clb+O in elderly or unfit pts with previously untreated CLL. Most pts with uMRD in the Ibr+Ven arm, including those with uIGHV, achieved clearance below 10 -5, and deeper clearance in PB was mirrored in BM. In the Ibr+Ven arm, clinical relapse was infrequent during the first year off treatment for pts with known MRD status at EOT+3 (whether uMRD or detectable MRD), supported by largely sustained uMRD/MRD levels over the same period. Additional follow-up will be important to confirm these early results. Figure 1 Figure 1. Disclosures Munir: Janssen, Abbvie, AstraZeneca, Morphosys, Alexion, Gilead, Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen, Abbvie, AstraZeneca, Alexion, Apellis, Gilead, Novartis: Honoraria. Moreno: Abbvie, Janssen, AstraZeneca, Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie, Janssen, AstraZeneca: Speakers Bureau; Janssen, Abbvie: Research Funding. Owen: Abbvie, AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen, Roche, Merck, Gilead, Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees. Follows: Roche, Abbvie, Janssen, Takeda, Janpix: Consultancy. Benjamini: AbbVie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Janssens: Abbvie, Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Trael Grant, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy; Beigene, AstraZeneca: Consultancy, Speakers Bureau. Levin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Robak: AstraZeneca, Abbvie, Janssen, Octapharma, Gilead,Oncopeptides AB, Pharmacyclics, Pfizer, GlaxoSmithKline, Biogen: Research Funding; Biogen, Abbvie, Octapharma, Janssen: Honoraria, Other: Advisory board; Medical University of Lodz: Current Employment. Simkovic: Janssen-Claig, Gilead, Roche, AstraZeneca, Abbvie: Consultancy, Honoraria, Other: Travel Grants, advisiory boards. Voloshin: Janssen, Abbvie, Sanofi, AstraZeneca, Takeda: Other: Clinical Trials, Non-finanfial support, Speakers Bureau; Novartis, Pfizer, MSD, La ROche: Other: Clinical Trials, Non-finanfial support. Vorobyev: Janssen, Roche, Sanofi, Takeda, Biocad, Abbvie: Other: Advisory Boards, Speakers Bureau; Astellas, Novartis, AstraZeneca: Speakers Bureau. Ysebaert: Abbvie, AstraZeneca, Janssen, Roche: Other: Advisory Board, Research Funding. Qi: Janssen: Current Employment. Steele: Janssen: Current Employment. Schuier: Janssen: Current Employment. Baeten: Janssen: Current Employment. Bennett Caces: Janssen: Current Employment. Niemann: Novo Nordisk Foundation: Research Funding; CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding. Kater: Abbvie: Honoraria, Other: Ad Board, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.

Abstract: The emergence of B cell receptor (BCR) kinase inhibitors has proved effective for the treatment of a number of B-cell malignancies including chronic lymphocytic leukemia (CLL). BTK and PI3K inhibitors have clear efficacy in suppressing tumor progression but have not been curative. A number of patients have developed resistance to these drugs following mutation of the BTK or PLCγ2 gene. Whilst, other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. Thus the development of novel drugs which are still effective once other BCR-kinases inhibitors become ineffective is of paramount importance. Spleen tyrosine kinase (Syk) is essential for B cell receptor signalling pathways as well as a variety of other surface receptors such as MHCII, FC receptors and integrins, all of which have been shown to play a role in CLL biology. Importantly, Syk inhibition has been shown to overcome resistance to ibrutinib, identifying Syk inhibition as a promising strategy to treat these patients. Furthermore, we have previously shown that IL-4 is found in CLL lymph nodes and can promote resistance to ibrutinib and idelalisib by restoring αIgM induced calcium flux and phosphorylated ERK (ASH 2014, abstract #3299). IL-4 signalling is mediated through the JAK/STAT signalling pathways via JAK1 and JAK3, therefore simultaneous inhibition of both Syk and JAK1/3 may be therapeutically beneficial over BCR kinase inhibitors alone. Cerdulatinib (PRT062070) is a dual JAK/Syk inhibitor in a phase I open label dose escalation study and is currently demonstrating clinical activity in patients with relapsed/refractory B cell malignancies including CLL. Our group has now demonstrated in vitro that cerdulatinib, at plasma concentrations achievable in patients, can induce apoptosis of CLL cells in a concentration and time dependent manner with a mean IC50 of 3µM and 1µM at 48 and 72h respectively, defined by annexin V/PI and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Apoptosis was caspase dependent since treatment with the pan caspase inhibitor ZVAD.fmk significantly inhibited cerdulatinib induced cell death at 24h. Cerdulatinib induced apoptosis coincided with an increase in pro-apoptotic proteins Noxa and Puma and a decrease in the anti-apoptotic protein Mcl-1. Cerdulatinib significantly inhibited IL-4 induced phosphorylation of STAT6 at 300nM (p=.005), BCR induced phosphorylation of AKTS473 with soluble (p=.008) and bead immobilised (BI) (p=.025) αIgM at 30nM and phosphorylation of AKTT308 with BI αIgM at 300nM (p=.008). Furthermore, in patients with CLL, it is thought that CD40L and IL-4 are key factors, which promote survival of CLL cells in proliferation centres within the lymph node microenvironment. Therefore, we cultured CLL cells with a vehicle control or IL-4\CD40L, prior to treatment with cerdulatinib. Cerdulatinib alone induced similar levels of apoptosis irrespective of IL-4/CD40L treatment, suggesting cerdulatinib may be able to overcome microenvironmental signals and target cells within the lymph node. Next we explored the possibility of augmenting cerdulatinib induced apoptosis by simultaneous inhibition with the Bcl-2\Bcl-XL inhibitor ABT-199. In vitro in the presence of IL-4/CD40L, ABT-199 synergised with cerdulatinib to induce significantly greater cell death than with either agent alone. Therefore these data provide in vitro evidence for the use of cerdulatinib in clinical trials for the treatment of CLL as either a single agent or in combination with other therapies such as ABT-199. Disclosures Strefford: Roche: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Coffey:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

Abstract: We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 μM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.