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Ibrutinib is associated with durable responses in patients with Waldenstrom macroglobulinaemia (WM). We hypothesized that response depth is predictive of progression-free survival (PFS) in WM patients treated with ibrutinib. Using landmark analyses, we evaluated response depth in two cohorts of WM patients treated with ibrutinib monotherapy. The learning cohort was composed of 93 participants from two clinical trials, and the validation cohort of 190 consecutive patients treated off clinical trial. Rates of partial response (PR) or better at six months in learning and validation cohorts were 64% and 71% respectively (P = 0·29). In the learning cohort, three-year PFS rates for patients who attained PR or better at six months versus not were 81% and 57% respectively (P = 0·009). In the validation cohort, three-year PFS rates for patients who attained PR or better at six months versus not were 83% and 54% respectively (P = 0·008). In multivariate analyses, attaining PR or better at six months was associated with superior PFS in the learning [hazard ratio (HR) 0·38; P = 0·01] and validation cohorts (HR 0·18; P = 0·004). Attaining PR at six months on ibrutinib emerges as an intermediate outcome of interest and should be validated as surrogate for PFS in clinical trials evaluating Bruton tyrosine kinase inhibitors in WM.

Activating MYD88 mutations promote pro-survival signaling through BTK and HCK, both targets of ibrutinib. Despite high response rates, complete responses to ibrutinib are lacking, and other MYD88 triggered pro-survival pathways may contribute to primary drug resistance. B-cell receptor (BCR) signaling has been observed in lymphomas driven by mutated MYD88, even without activating the BCR pathway mutations. We identified activated SYK (p-SYK), a component of BCR in complex with MYD88 in MYD88-mutated WM and ABC DLBCL lymphoma cells. Confocal microscopy confirmed co-localization of MYD88 with SYK in MYD88-mutated cells. Knockdown of MYD88 or use of a MYD88 signaling inhibitor abrogated SYK activation, while expression of mutated but not wild-type MYD88 amplified p-SYK in MYD88-mutated and wild-type lymphoma cells. Knockdown of SYK or use of inhibitors targeting SYK blocked p-STAT3 and p-AKT signaling in MYD88-mutated cells. Cell viability analysis showed that combining ibrutinib and SYK inhibitors triggered synthetic killing of MYD88-mutated lymphoma cells. Our findings extend the spectrum of mutated MYD88 pro-survival signaling to include SYK directed BCR cross talk in MYD88-mutated lymphomas. Targeting SYK in combination with ibrutinib produces synthetic lethality, providing a framework for the clinical investigation of ibrutinib with SYK inhibitors in MYD88-mutated lymphomas.

MYD88 mutations are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of Diffuse Large B-cell Lymphoma (ABC DLBCL). Mutated MYD88 activates BTK, and triggers downstream pro-survival signaling that includes NF-kB and ERK (Yang et al, Blood 2013; Blood 2016). ERK related signaling triggers inflammatory cytokine production including IL-6 and IL-10 (Chen et al, Blood 2016). Ibrutinib covalently binds to BTK Cys481 and inactivates BTK and downstream NF-kB and ERK signaling. Ibrutinib is approved for the treatment of WM and is associated with high overall response rates (>90%) and long term progression free survival in WM though intolerance to therapy, as well as resistance related to acquired BTK Cys481 mutations frequently leads to treatment discontinuation. We therefore investigated a novel, non-covalent BTK-inhibitor, pirtobrutinib that binds to BTK at non-Cys481 amino acids (G473-K483). Pirtobrutinib showed highly selective anti-proliferative activity against MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8 and HBL-1) versus MYD88 wild-type (OCI-Ly7, OCI-Ly19, Ramos, and RPMI-8226) cells, with marked apoptotic effect exhibited against primary MYD88 mutated WM cells at pharmacologically achievable levels (100-500 nM). Importantly, pirtobrutinib blocked BTK activity and overcame ibrutinib resistance in BCWM.1 WM and TMD-8 ABC DLBCL cells transduced to express both wild-type and mutated BTK (BTK Cys481Ser) with similar efficacy. The downstream signaling consequences of pirtobrutinib in vector only, wild-type and mutant BTK Cys481 expressing BCWM.1, MWCL-1, TMD-8 and HBL-1 cells was also examined. Treatment of vector only and wild-type BTK Cys481 expressing WM and ABC DLBCL cells with ibrutinib or pirtobrutinib abrogated both p-BTK and p-ERK signaling. In contrast, only pirtobrutinib blocked p-BTK and p-ERK signaling in mutant BTK Cys481 expressing WM and ABC DLBCL cells. In previous studies, we showed that inflammatory cytokine production that included IL-6 and IL-10 driven by ERK triggered ibrutinib resistance in wild-type BTK Cys481 MYD88 mutated lymphoma cells co-cultured with their mutated BTK expressing counterparts (Chen et al, BLOOD 2018). ERK-driven cytokine resistance to ibrutinib was postulated to explain how disease progression occurs in patients with modest variant expression of mutated BTK Cys481 (Woyach et al, JCO 2017; Xu et al, Blood 2017). Co-culture of BTK Cys481 mutated expressing TMD-8 cells with wild-type BTK expressing TMD-8 cells triggered resistance of the latter to ibrutinib. Treatment with pirtobrutinib blocked IL-6 and IL-10 production and overcame the protective effects conferred by BTK Cys481 mutated TMD-8 cells in these experiments. Lastly, oral administration of pirtobrutinib blocked p-BTK and p-ERK in BTK Cys481 mutated TMD-8 tumors xenografted in mice. Our findings therefore show that pirtobrutinib inhibits growth of MYD88 mutated lymphoma cells in a highly selective manner and can trigger apoptosis of primary WM patient BM lymphoplasmacytic cells at levels comparable to ibrutinib. Moreover, pirtobrutinib effectively blocked mutated BTK Cys481 driven BTK and ERK1/2 activation and produced similar cellular efficacy in both BTK wild-type and BTK Cys481 mutated cells. Pirtobrutinib also blocked the protective effect conferred to BTK wild-type cells through paracrine cytokines released by BTK Cys481Ser expressing cells. Lastly, pirtobrutinib blocked BTK and ERK1/2 activation in TMD8-BTK Cys481Ser xenografted mice. The findings support the development of pirtobrutinib in MYD88 driven lymphomas, including those resistant to ibrutinib on the bases of BTK Cys481 mutations. Disclosures Branagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

Ibrutinib is highly active in Waldenstrom macroglobulinaemia (WM) patients, but disease progression can occur due to acquired mutations in BTK, the target of ibrutinib, or PLCG2, the protein downstream of BTK. However, not all resistant patients harbour these alterations. We have performed a whole-exome sequencing study to identify alternative molecular mechanisms that can drive ibrutinib resistance. Our findings include deletions on chromosomes 6q, including homozygous deletions, and 8p, which encompass key regulators of BTK, MYD88/NF-κB, and apoptotic signalling. Moreover, we have identified recurring mutations in ubiquitin ligases, innate immune signalling, and TLR/MYD88 pathway regulators in ibrutinib-resistant WM patients.

Waldenstrom’s Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by bone marrow (BM) infiltration of immunoglobulin M (IgM)-secreting lymphoplasmacytic cells.[1][1] Activating mutations in MYD88 are present in 93-97% of WM and 50-70% of IgM monoclonal gammopathy of undetermined

CXCR4(WHIM) somatic mutations are common Waldenstrom's Macroglobulinemia (WM), and are associated with clinical resistance to ibrutinib. We engineered WM cells to express the most common WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis), CXCR(S338X) mutation in WM. Following SDF-1a stimulation, CXCR4(S338X) WM cells exhibited decreased receptor internalization, enhanced and sustained AKT kinase (AKT) and extracellular regulated kinase (ERK) signaling, decreased poly (ADP-ribose) polymerase and caspase 3 cleavage, and decreased Annexin V staining versus CXCR4 wild-type (WT) cells. CXCR4(S338X)-related signaling and survival effects were blocked by the CXCR4 inhibitor AMD3100. SDF-1a-treated CXCR4(S338X) WM cells showed sustained AKT and ERK activation and decreased apoptotic changes versus CXCR4(WT) cells following ibrutinib treatment, findings which were also reversed by AMD3100. AKT or ERK antagonists restored ibrutinib-triggered apoptotic changes in SDF-1a-treated CXCR4(S338X) WM cells demonstrating their role in SDF-1a-mediated ibrutinib resistance. Enhanced bone marrow pAKT staining was also evident in CXCR4(WHIM) versus CXCR4(WT) WM patients, and remained active despite ibrutinib therapy in CXCR4(WHIM) patients. Last, CXCR4(S338X) WM cells showed varying levels of resistance to other WM relevant therapeutics, including bendamustine, fludarabine, bortezomib and idelalisib in the presence of SDF-1a. These studies demonstrate a functional role for CXCR4(WHIM) mutations, and provide a framework for investigation of CXCR4 inhibitors in WM.

Introduction: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is the first approved therapy for Waldenström's macroglobulinemia (WM), and is highly active in both treatment-naive and relapsing or refractory patients. Although ibrutinib is highly active in WM patients, disease progression can occur. Acquired mutations in BTK at the binding site of ibrutinib (Cys481), or in the protein immediately downstream of BTK, the phospholipase PLCG2, have been identified in half of progressing WM patients on ibrutinib (Xu et al, Blood 2017). However, not all ibrutinib resistant patients harbor these alterations, suggesting that there are other causes of disease progression on ibrutinib. The aim of this study was to identify alternative molecular mechanisms that can drive ibrutinib resistance. Patients and Methods: Five WM patients who progressed while on ibrutinib were studied. Tumor DNA samples at diagnosis, relapse, and germline DNA were available in three patients. For the remaining two, relapse and germline samples were sequenced. DNA was extracted from CD19-selected bone marrow mononuclear cells from patients. Non-CD19 cells from peripheral blood were used as germline controls. Samples were sequenced using whole exome sequencing. Data were analyzed following the Broad Institute's GATK Best Practice Guidelines. Small variants were analyzed using Strelka and MuTect2. Somatic structural variants were detected using Manta, and copy number alterations were called using Control-FREEC. Results: Copy number analysis identified deletions in chromosome 6q in all patients, becoming homozygous in two of them at relapse. In another patient, the homozygous deletion was already present at baseline in a third of the tumor population, and increased at relapse. No other recurrent copy number alterations were detected, though in two patients multiple deletions or gains involving large chromosomal regions were observed. Regarding small variants, relapse samples showed a high proportion of acquired mutations detected at relapse only (median 85%, range 79%-88%) compared to persistent mutations detected at both baseline and relapse (median 15%, range 12%-21%). Three out of the five patients harbored mutations in BTK (two with p.Cys481Ser in the kinase domain and the other a p.Thr62Asn in the PH domain). In patients with a BTK mutation, other alterations were observed in genes related to the B-cell receptor pathway including PLCG2 p.Y495H; CD79B p.D33Y; LYN p.A2Stop and LYN p.A139T. In patients without BTK mutations, we identified several mutations with a putative role in ibrutinib resistance that emerged at relapse including AIP4, an E3 protein-ubiquitin ligase whose substrates are CXCR4, LYN or SYK, in which a recurring p.A646S mutation was observed in two patients; RNF19B, an E3 protein-ubiquitin ligase involved in the cytolytic activity of natural killer cells and cytotoxic T-cells, in which a p.R30G mutation was observed in two patients; FCRL3, an Fc receptor-like 3 that differentially modulates innate immune signaling in B cells, in which a p.E694Q was observed in one patient; Mutations in the negative regulators of Toll-like receptors signaling were observed that included DOK2 p.Y345Stop, and TOLLIP p.M242R and p.R228H which were each observed once in separate patients. Mutant allele frequency clustering analysis by k-means reflected a linear pattern of evolution from baseline to relapse, in which most persistent alterations maintained the same allele frequency (median 18%), and acquired mutations were present in a small proportion of tumor cells (median 9.3%, p Conclusions: Our findings depict uniform deletion of 6q, including homozygous loss of 6q, which encompass key regulators of BTK, BCL2, and NFKB; as well as emergence of novel gene mutations, including recurring mutations in E3 ubiquitin ligases, innate immune signaling, and Toll receptor/MYD88 pathway regulators as significant genomic alterations that accompany disease progression on ibrutinib in WM patients. Disclosures Castillo: Genentech: Consultancy; Beigene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; BMS: Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.

Background Mutations in MYD88 are highly recurring in Waldenstrom's Macroglobulinemia (WM) patients and are important for establishing the diagnosis of WM. The most common mutation in MYD88 is c.978T>C resulting a proline substitution for leucine at amino acid position 265 (p.Leu265Pro). Both allele specific PCR (AS-PCR) and clinical diagnostic next generation sequencing (NGS) panels are used to detect mutated MYD88, though they differ in sensitivity and scope. In this study we screened 734 patients with WM by AS-PCR for MYD88 c.978T>C MYD88 followed by Sanger sequencing to clarify negative results for non-MYD88 p.Leu265Pro mutations and compared the findings to clinical NGS panel data from the same biopsy when available. We also investigated MYD88 isoform dysregulation and isoform specific effects of the observed mutations that may impact mutated MYD88 regulation which has not been previously studied in WM. Methods DNA from CD19-selected bone marrow mononuclear cells (BMMC) of 734 WM patients were used for the MYD88 c.978T>C AS-PCR assay previously described by us (Xu et al, Blood 2013). For patients wild-type for MYD88 c.978T>C by AS-PCR, Sanger sequencing of the open reading frame of MYD88 was performed for both DNA and RNA simultaneously isolated from CD19-selected BMMC. DNA was also used to validate the presence of c.978T>C by Sanger. Findings were compared to 222/734 (30.2%) patients who also underwent illumina miSeq based targeted next generation sequencing on a clinical diagnostic platform using unselected BMMC. NGS isoform specific expression estimates were calculated using Salmon for 77 WM patients and 34 healthy donors (Hunter et al, Blood 2016). Results 688/734 (93.7%) WM patients tested positive for the c.978T>C mutation. To confirm these results, Sanger sequencing at the DNA level covering the c.978T>C mutation was performed in 361/688 (52.5%) patients confirming the presence of the mutation in all cases. These Sanger studies revealed that one patient had two somatic mutations in addition to c.978T>C. Of the 46/734 (6.3%) that were wild-type by AS-PCR, 18 had cDNA available to screen for alternative MYD88 mutations. Of these, 13/18 (72.2%) were confirmed to be truly wild type for MYD88, and 5/18 (27.8%) harbored alternative MYD88 mutations making up 0.7% of the study population. Taken together 693/734 (94.4%) of patients were found to harbor somatic MYD88 mutations. Of the 222 patients form whom matching NGS panel data was available, the finding between the NGS and AS-PCR studies were largely concordant. The only discrepancies observed were 69 (31.1%) cases where targeted NGS gave false negative results for c.978T>C but was detected by AS-PCR. Of the four patients with alternative MYD88 mutations, one patient had a dinucleotide substitution that also resulted in p.Leu265Pro but tested as wild-type by AS-PCR, two patients each had one previously documented mutation (either pVal217Phe or p.Ser243Asn) and one patient had a mutation that was synonymous at the protein level (p.Phe277Phe). The patient with the two novel mutations in addition to c.978T>C had a mutation in the polypyrimidine track leading to the final exon and one resulting in p.Gly259Gly in the primary transcript but presents as a highly disruptive p.Val199Glu in the shorter regulatory isoforms. This is similar to c.978T>C which presents as p.L265P in the primary transcripts but acts as a stop loss in the shorter isoforms. We therefore looked for evidence of isoform level dysregulation in MYD88 using RNASeq and found highly significant and distinctive MYD88 isoform signatures for MYD88 mutant, MYD88 wild-type and healthy donor samples (Figure 1). Conclusions Using CD19-selected BMMC, MYD88 c.978T>C (p.Leu265Pro) was found in 93.7% of 734 patients, while non-c.978T>C mutations were present in Figure 1. Figure 1. Disclosures Castillo: Millennium: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding. Treon:Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; BMS: Research Funding; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Hunter:Pharmacyclics: Consultancy.

Activating mutations in MYD88, a component of the Toll-receptor (TLR) pathway, trigger Myddosome self-assembly and multiple downstream pro-survival signaling through BTK and IRAK4/IRAK1 (Yang et al, Blood 2013). Activation of B-cell receptor (BCR) signaling has also been observed in WM, though the mechanism(s) remain to be clarified (Argyropoulos et al, Leukemia 2016). While activating mutations in the BCR components CD79A/B are common in MYD88 mutated ABC DLBCL, they are uncommon in WM (Ngo et al, Nature 2011; Hunter et al, Blood 2014). We therefore sought to clarify if TLR/MYD88 crosstalk could explain aberrant BCR signaling in WM. We performed PhosFlow analysis of MYD88 and BCR signaling components of MYD88 mutated and wild-type WM and ABC DLBCL cell lines. These studies showed high levels for expression of the BCR component p-SYK (Y525-526) in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cell lines versus MYD88 wild-type cell lines. High levels of p-SYK were also observed in primary MYD88 mutated WM cells compared to healthy donor peripheral blood B-cells. Following treatment with a MYD88 blocking peptide, p-SYK was robustly reduced in WM cell lines, while only modest reduction was observed in ABC DLBCL cell lines which also carry activating CD79B mutations. Use of MYD88 inhibitor also blocked the p-SYK in primary MYD88 mutated WM cells. Lentiviral over-expression of MYD88 L265P but not wild-type MYD88 or vector control augmented p-SYK expression in MYD88 mutated BCWM.1 WM cells, as well as MYD88 wild-type Ramos and OCI-Ly7 cells. Conversely, knockdown of MYD88 in BCWM.1 cells confirmed the dependence of p-SYK on mutated MYD88. p-SYK was also confirmed to be in complex with the Myddosome in BCWM.1 cells by co-immunoprecipitation (co-IP) using anti-MYD88 and anti-SYK/anti-p-SYK specific antibodies. Over-expression and knockdown studies of mutated MYD88 also showed an association of downstream p-STAT3 (Y705) signaling on MYD88 triggered p-SYK, a finding confirmed by use of SYK inhibitors which blocked p-STAT3 in a dose-dependent manner. CellTiter-Glo® viability assays showed the SYK inhibitors, R406 and Entospletinib (GS-9973), produced higher cytotoxicity in MYD88 mutated versus wild-type B-cell lines. (Figure 1A) Since BCR/SYK kinase triggers divergent signaling from TLR/BTK/IRAK pathways, we examined the combined effect of BTK and SYK inhibition. Combination index and normalized isobologram analysis demonstrated synergistic effects with the combination of ibrutinib with either R406 or Entospletinib in MYD88 mutated WM and ABC DLBCL cell lines (Figure 1B, 1C). The combination of ibrutinib with either R406 or Entospletinib also produced more robust tumor cell apoptosis in primary MYD88 mutated WM cells. Our findings show that MYD88 can cross-talk with BCR pathway through SYK tyrosine kinase, and trigger aberrant p-STAT3 signaling. The inhibition of both TLR/BTK and BCR/SYK activation by use of ibrutinib and SYK inhibitors produced synergistic tumor cell killing of MYD88 mutated WM and ABC DLBCL lymphomas, and provides a framework for clinical studies aimed at extinguishing aberrant MYD88 signaling. Disclosures Castillo: Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Hunter:Pharmacyclics: Consultancy. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

Activating mutations in MYD88 are present in many B-cell lymphoproliferative disorders including WM (95%), ABC DLBCL (39%), Primary CNS Lymphoma (80-90%), Marginal Zone Lymphoma (6-10%) and Chronic Lymphocytic Leukemia (4-8%). In MYD88 mutated WM and ABC DLBCL cells, MYD88 triggers NF-kB pro-growth and survival signaling through divergent pathways driven by BTK and IRAK4/IRAK1 (Ngo et al, Nature 2011; Yang et al, Blood 2013). Ibrutinib targets BTK, and has shown high levels of activity in MYD88 mutated WM, ABC DLBCL and PCNSL. Responses however are partial, and complete remissions are lacking. We therefore sought to clarify if persistent IRAK4/IRAK1 signaling was responsible for survival of malignant lymphoplasmacytic cells (LPC) in WM patients on ibrutinib. We observed by flow cytometric analysis that while BTK activation was suppressed in WM patients on ibrutinib for >6 months, both IRAK4 and IRAK 1 remained hyperactivated. Treatment of LPC from patients on ibrutinib with a toolbox IRAK4/IRAK1 inhibitor resulted in marked reduction of NF-kB and induction of apoptosis. Subsequently, we performed lentiviral transduction studies in MYD88 mutated BCWM.1 cells, and observed in an inducible model system that knockdown of IRAK1 produced more robust apoptotic effects versus IRAK4 (Figure 1A). Given these findings, we have pursued a medical chemistry campaign to develop a potent and highly selective inhibitor of IRAK1 kinase activity, JH-X-119-01. JH-X-119-01 inhibited IRAK1 biochemically with an IC50 of 9.3 nM while exhibiting no inhibition of IRAK4 at concentrations up to 10 µM, and showed exceptional Kinome selectivity with off-target inhibition of only two kinases, YSK4 and MEK3 (Figure 1B). Mass spec labelling studies were used to confirm that JH-X-119-01 irreversibly labelled IRAK1 at cysteine 302. JH-X-119-01 showed antiproliferative effects on MYD88 mutated WM and ABC DLBCL cells. Importantly, the combination of JH-X-119-01 with Ibrutinib led to synergistic tumor cell killing in MYD88 mutated WM and ABC-DLBCL cells, and suppression of NF-ĸB activation (Figure 1C). In vivo PK studies revealed a favorable profile for JH-X-119-01 with a moderate half-life of 1.61 hours, a Cmax of 9.95 µM, and a low clearance of 18.84 mL/min/kg when dosed IV. Our findings evidence the development of a novel, highly selective IRAK1 kinase inhibitor, JH-X-119-01, that shows specific inhibition of IRAK1 kinase activity and a reduction of tumor cell survival in MYD88 mutated WM. Importantly, JH-X-119-01 shows synergistic tumor cell killing with ibrutinib in MYD88 mutated WM and ABC-DLBCL cells. This study provides a framework for the development of highly selective IRAK1 inhibitors for use alone, and in combination with ibrutinib in MYD88 mutated B-cell lymphomas. Figure 1 Figure 1. Disclosures Castillo: Janssen: Consultancy, Research Funding; Abbvie: Research Funding; Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding. Treon: Pharmacyclics: Consultancy, Research Funding.

Waldenstrom's Macroglobulinemia (WM) is characterized by bone marrow (BM) involvement of IgM secreting lymphoplasmacytic lymphoma. Whole genome sequencing identified somatic activating mutations in MYD88 and CXCR4 in 95% and 35-40% of WM patients, respectively. Somatic mutations were observed in ARID1A, CD79B, TP53, and MYBPB1A in MYD88 mutated WM patients. Deletions in in chromosome 6q were also observed in half of MYD88 mutated patients. The somatic events that drive MYD88 wild-type (WT) WM remain to be defined. Patients with MYD88 WT WM show an increased risk of death compared to their MYD88 mutated counterparts (Treon et al, Blood 2014). Moreover, WM patients with MYD88 WT disease show no major response to ibrutinib versus 81% in MYD88 mutated patients (Treon et al, NEJM 2015). These findings point to fundamental genomic differences in tumor biology between MYD88 mutated and wild-type patients. To better understand this rare and aggressive form of WM, we performed whole exome sequencing of 12 WM patients who met the clinicopathological diagnostic criteria for WM using WHO and WM consensus criteria. These patients were WT for MYD88 following genotyping by highly specific AS-PCR for MYD88 L265P and Sanger sequencing to exclude non-L265P MYD88 mutations. CD19-selected BM mononuclear cells were used for tumor sampling, and CD19-depleted cells for germline comparisons. This cohort was split evenly between males and females. Their median age was 58 (range 50-85 years), median BM disease involvement was 12.5% (range 2.5%-80%), and median serum IgM was 4,055 (range 1,070-5,620 mg/dL). Seven (58%) patients had adenopathy, and 3 (25%) had splenomegaly. Seven (58%) patients were previously treated. Next generation sequencing was performed by the Center for Cancer Computational Biology, and the data were analyzed following the Broad Institutes GATK Best Practice Guidelines. Small variants were analyzed using both Strelka and MuTect2. Somatic structural variants were detected using Manta, and copy number alterations were called using Control-FREEC. All somatic variants were validated by Sanger sequencing. The most recurrently mutated gene was TBL1XR1, which was observed in 3 (25%) patients, with one patient harboring two TBL1XR1 mutations. Two missense mutations were observed within distinct WD40 domains, while an early frameshift mutation is predicted to impact all WD40 domains. A fourth mutation is predicting to effect splicing. TBL1XR1 mutants in the WD40 domain are found in marginal zone lymphomas and are known to increase TBL1XR1 binding with nuclear receptor corepressor (NCoR), leading to increased degradation of NCoR and the activation of NF-κB and JUN target genes (Jung et al, Oncotarget 2017). Novel, truncating MALT1 mutations were present in two (17%) patients. Notably, these nonsense mutations were 23 base pairs apart and predicted for truncation of the c-terminal domain with loss of a TRAF6 binding site. Finally, a BCL10 nonsense mutation was observed just upstream of a critical regulatory phospho-serine site. Both MALT1 and BCL10 are members of the CARD11-BCL10- MALT1 (CBM) complex that signals downstream to NF-kB. Other validated somatic mutations included TIMELESS (n=3; 25%), SHROOM3 (n=2; 17%), BLM (n=1; two mutations found in the same patient); KCNB1 (n=1), IGF1-R (n=1), NOTCH1 (n=1). No CXCR4 mutations were detected. The genomic and protein coordinates for these somatic mutations are shown in Table 1. Our structural variant analysis also identified large deletions that removed the DEATH/PEST domain of NFKB2 in two patients covering amino acids 691-822 and 711- 839, respectively. Deletions in the DEATH/PEST domain of NFKB2 are associated with constitutive non-canonical NF-kB activation. All together, NF-kB related mutations were present in 6/12 (50%) MYD88 wild-type WM patients. Analysis of copy number alterations revealed no highly recurrent events, but was remarkable for the absence of deletions on chromosome 6q which are typically found in 50% of MYD88 mutated WM patients. Our findings highlight for the first time the genomic landscape that surrounds MYD88 wild-type WM, and point to a distinct mutational profile from MYD88 mutated WM. The findings also include several novel somatic mutations not previously reported in other B-cell lymphoproliferative disorders. Table 1 Table 1. Disclosures Castillo: Pharmacyclics: Consultancy, Research Funding; Millennium: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding. Treon: Pharmacyclics: Consultancy, Research Funding.

Activating mutations in MYD88 promote NF-kB survival signaling in WM and ABC DLBCL cells through BTK and HCK, both targets of ibrutinib (Yang et al, Blood 2013; 2016). These findings likely explain the high rates of response observed in MYD88 mutated WM and ABC DLBCL patients (Treon et al, NEJM 2015; Wilson et al, Nature Med 2015). Resistance to ibrutinib is increasingly being recognized with prolonged therapy in patients with various B-cell malignancies. Mutations at the BTKCys481 site are observed in half of WM patients with disease progression following initial response (Xu et al, Blood 2017), and are also associated with clinical resistance in patients with CLL and MCL. However, in many WM and CLL cases, BTKCys481 mutations are subclonal and their relevance to clinical disease progression remains unclear. Moreover, the signaling mechanisms that promote ibrutinib resistance remain to be clarified. We therefore performed lentiviral transduction studies in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cells with vector alone, wild-type BTK (BTKWT), and the BTK variant most frequently observed in ibrutinib resistant WM and CLL cases (BTKCys481Ser). BTKCys481Ser but not BTKWT or vector only transduced WM and ABC DLBCL cells uniformly demonstrated persistent BTK and PLCγ2 activation in the presence of ibrutinib (100, 500 nM), along with a 1-3 log fold increase in EC50 to ibrutinib. Western blot analysis showed persistent BTK signaling in BTKCys481Ser expressing cells that was accompanied by ERK1/2 hyperactivation. Use of highly selective and potent ERK1/2 inhibitors (ulixertinib, GDC-0994) induced apoptosis of BTKCys481Ser expressing WM and ABC DLBCL cells, and showed synergistic tumor cell killing with ibrutinib by CellTiter-Glo® Luminescent Cell Viability Assays using Calcasyn, as well as enhanced apoptosis by Annexin V staining. Using a multiplex cytokine assay and confirmed by TaqMan® quantitative RT-PCR assay for cytokine mRNA levels, we identified that ERK1/2 activation in ibrutinib treated BTKCys481Ser WM and ABC DLBCL cells was accompanied by persistent release of pro-inflammatory and growth enhancing cytokines including CXCL13, IL-2R, IL-6, IL-10, IL-12p40, and TNF-a that was blocked by ulixertinib. In contrast, ibrutinib blocked their release in BTKWT WM and ABC DLBCL cells. We next sought to clarify if cytokine release by ibrutinib treated BTKCys481Ser WM and ABC DLBCL could impact survival of bystander tumor cells. We performed experiments using a Transwell system in which BTKCys481Ser or BTKWT transduced WM or ABC DLBCL cells were co-cultured with their native counterpart cells in the presence or absence of ibrutinib (Figures 1A, B). These studies showed that native WM or ABC DLBCL cells were rescued in the presence of ibrutinib when co-cultured with their BTKCys481Ser but not BTKWT transduced counterparts. Finally, use of IL-6 and IL-10 blocking antibodies abolished the protective effect on native tumor cells conferred by co-culture with BTKCys481Ser expressing cells in the presence of ibrutinib (Figures 1C, D). Our findings show that BTKCys481Ser mutation drives ibrutinib resistance through hyperactivation of ERK1/2, and that paracrine mediated ERK1/2 dependent pro-survival signaling by BTKCys481Ser expressing cells can confer a protective effect on bystander Waldenstrom's Macroglobulinemia and ABC DLBCL cells in the presence of ibrutinib. Figure 1 Figure 1. Disclosures Castillo: Janssen: Consultancy, Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Research Funding; Millennium: Research Funding. Treon: Pharmacyclics: Consultancy, Research Funding.

CXCR4(WHIM) frameshift and nonsense mutations follow MYD88(L265P) as the most common somatic variants in Waldenstrom Macroglobulinaemia (WM), and impact clinical presentation and ibrutinib response. While the nonsense (CXCR4(S338X) ) mutation has been investigated, little is known about CXCR4 frameshift (CXCR4(FS) ) mutations. We engineered WM cells to express CXCR4(FS) mutations present in patients, and compared their CXCL12 (SDF-1a) induced signalling and ibrutinib sensitivity to CXCR4(wild-type (WT)) and CXCR4(S338X) cells. Following CXCL12 stimulation, CXCR4(FS) and CXCR4(S338X) WM cells showed impaired CXCR4 receptor internalization, and enhanced AKT1 (also termed AKT) and MAPK1 (also termed ERK) activation versus CXCR(WT) cells (P < 0·05), though MAPK1 activation was more prolonged in CXCR4(S338X) cells (P < 0·05). CXCR4(FS) and CXCR4(S338X) cells, but not CXCR4(WT) cells, were rescued from ibrutinib-triggered apoptosis by CXCL12 that was reversed by AKT1, MAPK1 or CXCR4 antagonists. Treatment with an inhibitor that blocks MYD88(L265P) signalling triggered similar levels of apoptosis that was not abrogated by CXCL12 treatment in CXCR4(WT) and CXCR4(WHIM) cells. These studies show a functional role for CXCR4(FS) mutations in WM, and provide a framework for the investigation of CXCR4 antagonists with ibrutinib in CXCR4(WHIM) -mutated WM patients. Direct inhibition of MYD88(L265P) signalling overcomes CXCL12 triggered survival effects in CXCR4(WHIM) -mutated cells supporting a primary role for this survival pathway in WM.