ALBERT

Description: Introduction: Approximately 40% of patients with Waldenström macroglobulinemia (WM) have an activating somatic mutation in CXCR4, including both nonsense and frameshift variants. There are limited data on the impact of CXCR4 mutations on the outcomes to therapy in WM patients. Mounting evidence suggests that CXCR4 mutations adversely affect depth of response and progression-free survival (PFS) to ibrutinib in patients with WM. Methods: We performed a pooled analysis evaluating the impact of CXCR4 mutations and CXCR4 mutation subtypes on response, PFS and survival after frontline treatment initiation (SAFTI) on 76 WM patients who received proteasome inhibitor-based primary therapy. All patients were participants on three prospective clinical trials evaluating the combinations of bortezomib, dexamethasone and rituximab (BDR; ClinicalTrials.Gov ID NCT00250926), carfilzomib, dexamethasone and rituximab (CaRD; ClinicalTrials.Gov ID NCT01470196), and ixazomib, dexamethasone and rituximab (IDR; ClinicalTrials.Gov ID NCT02400437) in previously untreated patients with WM, and were treated at the Bing Center for WM. All patients met criteria for a clinicopathological diagnosis of WM and for treatment initiation, according to the guidelines established by the 2nd International Workshop for WM (IWWM). CXCR4 mutations were divided in nonsense and frameshift mutations, as previously described. Response to therapy was assessed using response criteria by the 3rdIWWM. We fitted univariate and multivariate logistic regression and proportional-hazard Cox regression models for major response and PFS and SAFTI, respectively. P-values

Description: Introduction: The Bruton tyrosine kinase inhibitor ibrutinib is the only FDA approved therapy for the treatment of symptomatic Waldenstrom macroglobulinemia (WM), and has been associated with high response rates and durable progression-free survival (PFS). Factors associated with depth of response and PFS duration are not well established. We performed a retrospective study aimed at identifying predictive and prognostic factors in WM patients treated with ibrutinib. Methods: We included consecutive patients with a diagnosis of WM treated with ibrutinib monotherapy evaluated at the Dana-Farber Cancer Institute since January 2012 through March 2019. Patients with Bing-Neel syndrome (WM involving the central nervous system) were excluded. Baseline clinical and laboratory characteristics were gathered. MYD88 and CXCR4 mutations were assessed using polymerase chain reaction assays and Sanger sequencing. Responses at 6 months were assessed using criteria from IWWM3. PFS was defined as the time from ibrutinib initiation until last follow-up, death or progression. Univariate and multivariate logistic regression models were fitted for partial response (PR) and very good partial response (VGPR) at 6 months, and Cox proportional-hazard regression models were fitted for PFS. Results: A total of 252 patients were included in our analysis. Selected baseline characteristics include: age ≥65 years (60%), hemoglobin

Description: Activating mutations in MYD88 alone or in coordination with BCR activating mutations transactivate Bruton's tyrosine kinase (BTK) in WM and ABC DLBCL cells (Yang et al, Blood 2013; Wilson WH et al, Nat Med 2015; Phelan JD, Nature 2018). Ibrutinib is a covalent inhibitor that binds to BTKCys481 and is active in MYD88 mutated WM and ABC-DLBCL (Treon et al, NEJM 2015; Wilson et al, Nat Med, 2015). Acquired resistance to ibrutinib is increasingly being recognized in WM, as well as other B-cell malignancies due to somatic mutations at BTKCys481 that abrogate BTK-ibrutinib binding (Xu et al, Blood 2017). BTKCys481 mutations are usually sub-clonal, but can drive ibrutinib resistance and protect BTK wild-type clones through a paracrine mechanism involving activation of ERK1/2 (Chen et al, Blood 2018). Hematopoietic cell kinase (HCK) which is aberrantly upregulated and transactivated by mutated MYD88, and triggers multiple pro-survival signaling cascades that include activation of BTK, as well as PI3K/AKT and MAPK/ERK1/2 (Yang G et al, Blood 2016). We therefore examined if inhibition of HCK could abrogate BTK activity and overcome ibrutinib resistance driven by BTKCys481 mutations in MYD88 mutated B-cell lymphomas. We performed a screen of 220 clinical and preclinical kinase inhibitors to identify compounds with potent HCK inhibition. Over 100 analogs of three series of promising compounds with HCK activity were synthesized and triaged. Target deconvolution was performed to clarify selectivity, and other important kinase targets. These efforts led to the selection of a lead compound identified as a type-II kinase inhibitor, KIN-8193, with a molecular weight around 600. Single-digit nanomolar (nM) biochemical and double-digit nM cellular potency was demonstrated, with high selectivity (S-score 0.07) in line with that observed with ibrutinib (S-score of 0.03). Live-cell target engagement for HCK was confirmed by competitive ATP-biotin binding assay. DMPK and PK studies showed very high levels of mouse, rat, and human microsomal stability (42.4, 60.2 and 79.3 minutes respectively), and oral bioavailability in mice (48%) and rats (79%). Cmax reached 2.0 µM, while T1/2 was 26.8 hours with 25 mg/kg single oral dosing in rats. Rats toxicology studies showed excellent tolerability in 28-days repeated oral dosing with 25 mg/kg/biw dosing schedule. No relevant inhibition was observed against a panel of 100 other receptor targets, including hERG. AMES was negative up to 100 uM, and Cyp inhibition studies showed acceptable inhibition up to 10 uM. KIN-8193 potently inhibited the phosphorylation of HCK(p-Y411) and its downstream target BTK(p-Y223) in both BTK wild-type and ibrutinib-resistant BTKCys481 mutated WM and ABC DLBCL cell lines driven by activating MYD88 mutations, and primary WM cells (Fig 1A). Target engagement studies showed HCK but not BTK direct binding. In vivo pharmacodynamic (PD) studies using luciferized BCWM.1 cells WM xenograft mouse model showed potent dose-dependent inhibition of HCK(p-Y411) and BTK(p-Y223) at 6 and 24 hours following single dose administration of KIN-8193. Importantly, KIN-8193 showed selective cytotoxicity against MYD88 mutated BTK wild-type and BTKCys481 mutated, ibrutinib-resistant WM and ABC DLBCL cell lines, and primary WM cells, but had no impact on healthy donor B- and T-cells at pharmacologically achievable levels (Fig. 1B). We therefore describe a novel, highly selective and potent HCK inhibitor that is well tolerated in long-term rat toxicology studies and shows selective killing of MYD88 mutated WM and ABC DLBCL cells. Inhibition of HCK by KIN-8193 blocks downstream wild-type BTK and Cys481 mutated BTK activity, and overcomes ibrutinib resistance induced by BTKCys481 mutations. Disclosures Hunter: Pharmacyclics: Consultancy. Castillo:Genentech: Consultancy; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Millennium: Research Funding. 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.

Description: 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

Description: Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs) that is down-regulated in later stages of B-cell ontogeny. In MYD88 mutated B-cell lymphomas, HCK is aberrantly over-expressed and is activated and triggers multiple growth and survival pathways including BTK, PI3Kδ/AKT and ERK1/2 which are essential to tumor cell survival. Ibrutinib, a pleiotropic inhibitor of BTK, that produces remarkable responses in MYD88 mutated WM, ABC-DLBCL, and Primary CNS Lymphoma was found also potently inhibits HCK. Mutations that abolish ibrutinib-HCK binding greatly diminish anti-tumor activity in MYD88 mutated lymphoma cells, highlighting the importance of HCK as an essential target in MYD88 driven diseases. To clarify the transcriptional regulation of HCK in MYD88 mutated malignancies, we performed promoter binding TF profiling, PROMO weighted transcription factor (TF) consensus binding analysis, and chromatin immuno-precipitation (ChIP) studies. We identified PAX5, and mutated MYD88 downstream signaling mediators, STAT3, AP-1 and NF-kB as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88 mutated lymphoma cells. Deletion of STAT3, AP-1 or NF-kB binding sites greatly reduced corresponding TFs binding and HCK promoter activity, indicating the importance of MYD88 directed signaling in the regulation of HCK transcription. Among AP-1 complex components, JunB but not c-Jun showed greatest relevance to TLR/MYD88 signaling and regulation of HCK transcription. Since STAT3 and NF-kB are known downstream mediators of mutated MYD88 signaling, we focused on the function of JunB. JunB phosphorylation increased following MYD88 pathway activation through TLR4 (with LPS-EB) or TLR9 (with ODN-2006), as well as lentiviral mediated expression of mutated MYD88 (L265P) but not wild type MYD88 (MYD88-WT) at Thr102 and Thr104 in either MYD88 mutated BCWM.1 cells or MYD88-WT Ramos cells. Conversely, c-Jun phosphorylation was not impacted by either intervention. Knockdown of MYD88 in BCWM.1 WM cells also reduced the phosphorylation of JunB, while c-Jun phosphorylation showed modest increase. Moreover, knockdown of JunB reduced HCK protein levels in MYD88 mutated WM and ABC-DLBCL cells. These data demonstrate that JunB is an important mediator of mutated MYD88 signaling among AP1 complex members and is directly involved in the regulation of HCK expression in MYD88 mutated B-cell lymphoma. The findings provide new insights into the transcriptional regulation of HCK by MYD88 driven TFs, and opportunities for further advancing targeted therapeutics in MYD88 driven B-cell malignancies. Disclosures Hunter: Janssen: Consultancy. Castillo:TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

Description: 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.

Description: Background: Waldenstrom's Macroglobulinemia/IgM lyphoplasmacytic lymphoma is a B-cell lymphoma defined by highly recurring mutations in MYD88 (95-97%) and CXCR4 (30-40%) by whole genome, PCR and Sanger sequencing (Treon et al NEJM 2012, Hunter et al Blood 2013, Xu et al, BJH 2016). Using next generation RNA sequencing studies (RNASeq) of these same group of patients, we subsequently observed that MYD88 and CXCR4 mutation status were the primary determinants of differential gene expression in WM (Hunter et al, Blood 2016). We have now integrated enhanced reduced representation bisulfite sequencing (ERRBS) data into the existing WGS and RNASeq data to present of more complete picture of genomic regulation in WM. Methods: CD19+ selected bone marrow samples from 54 patients with WM, and CD19- peripheral blood mononuclear samples were used for tumor and germline controls, respectively. WGS and RNASeq were conducted as previously described (Hunter et al, Blood 2014; 2016) and methylation profiling was conducted using ERRBS at the Weil Cornell Medical Epigenomics Core and analyzed using Bismark. Results were filtered for methylation sites supported by at least 10 reads across all of the samples. Differential methylation analysis was conducted at the individual site level and aggregated promoter level using an established edgeR protocol. Differential methylation data was then compared with Salmon derived gene and isoform expression data which was available for 49/54 (91%) of the samples. Results: Clinical characteristics of patients were as follows: Median age 60 (range 40-87 years); BM involvement 43% (range 4-95%); serum IgM 3590 (range 416-8320 ug/dl). Most patients (67%) were untreated. WGS data revealed 33 (61%), 18 (33%), and 3 (6%) were MYD88Mutant/CXCR4Wild-Type, MYD88Mutant/CXCR4Mutant and MYD88Wild-Type/CXCR4Wild-Type, respectively. As was observed in our RNASeq analysis, pairwise multidimensional scaling of the methylation status of the top 2,000 high variance promoters revealed segregation of the MYD88Mutant/CXCR4Wild-Type from both MYD88Mutant/CXCR4Mutant and MYD88Wild-Type/CXCR4Wild-Type (Figure 1A). Log fold change in methylation in differentially methylated promoters from MYD88Mutant/CXCR4Mutant and MYD88Wild-Type/CXCR4Wild-Type samples relative to MYD88Mutant/CXCR4Wild-Type were negatively correlated with log-fold change in gene expression (Rho = -0.463, p

Description: 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

Description: Key Points BTKCys481 mutation results in ERK1/2 mediated survival signaling and ibrutinib resistance in MYD88-mutated cells. BTKCys481 mutation confers a protective effect against ibrutinib on neighboring BTK wild-type cells through a paracrine mechanism.

Description: Activating mutations in MYD88 promote malignant cell growth and survival through multiple pathways that include BTK and HCK. HCK is transcriptionally upregulated and activated by mutated MYD88 and in turn activates BTK itself, as well as ERK and AKT. Ibrutinib is a covalent inhibitor that binds to BTKCys481 and shows activity in MYD88 mutated B-cell malignancies, including WM, MZL, ABC DLBCL, and PCNSL. Resistance to ibrutinib on the basis of BTKCys481 as well as downstream mutations is increasingly being recognized. We therefore sought to develop potent and selective inhibitors that target HCK. We developed a non-covalent dual inhibitor, KIN-8194, of HCK and BTK following a screen 〉220 clinical and preclinical kinase inhibitors, and lead optimization following synthesis 〉400 analogs. Dual HCK and BTK inhibition was confirmed by both KINOMEscan® and live-cell target engagement studies (KiNativ™ profiling and live cell ATP-biotin competition assay). KIN-8194 showed robust suppression of HCK (IC50