“Double-Hit” Chronic Lymphocytic Leukemia, Involving the TP53 and MYC Genes

Although the 17p deletion [del(17p)] is rare in cases of treatment-naive chronic lymphocytic leukemia (CLL), its frequency is higher in refractory/relapsed CLL – particularly in patients undergoing chemo(immuno)therapy. TP53 disruption (deletion and/or mutation) is the strongest prognostic factor for refractoriness to chemotherapy; the use of Bruton tyrosine kinase inhibitors and BCL2 inhibitors is then indicated. Rare cases of CLL can also harbor translocation or gain of the MYC oncogene. “Double-hit CLL” (with del(17p) and MYC gain) is associated with a very poor prognosis. The prognostic impact of TP53 disruption with MYC aberrations in patients receiving targeted therapies must now be evaluated.

Histone neutralization in a rat model of acute lung injury induced by double-hit lipopolysaccharide

Background: Acute respiratory distress syndrome (ARDS) remains a challenge because of its high morbidity and mortality. Circulation histones levels in ARDS patients were correlated to disease severity and mortality. This study examined the impact of histone neutralization in a rat model of acute lung injury (ALI) induced by a lipopolysaccharide (LPS) double-hit. Methods: Sixty-eight male Sprague-Dawley rats were randomized to sham (N=8, received saline only) or LPS (N=60). The LPS double-hit consisted of a 0.8 mg/kg intraperitoneal injection followed after 16 hours by 5 mg/kg intra-tracheal nebulized LPS. The LPS group was then randomized into five groups: LPS only (N=12); LPS + 5, 25, or 100 mg/kg intravenous STC3141 every 8 hours (LPS+L, LPS+M, LPS+H, respectively, each N=12); or LPS + intraperitoneal dexamethasone 2.5 mg/kg every 24 hours for 56 hours (LPS+D, N=12) The animals were observed for 72 hours. Results: LPS animals developed ALI as suggested by lower oxygenation, lung edema formation, and histological changes compared to the sham animals. Compared to the LPS group, LPS+H and +D animals had significantly lower circulating histone levels; only the LPS+D group had significantly lower bronchoalveolar lavage fluid (BALF) histone concentrations. The LPS+L, +M, +H and +D groups had improved oxygenation compared to the LPS group and the LPS+H and +D groups had a lower lung wet-to-dry ratio. All animals survived. Conclusion: Neutralization of histone using STC3141, especially at high dose, had similar therapeutic effects to dexamethasone in this LPS double-hit rat ALI model, with significantly decreased circulating histone concentration, improved oxygenation, and decreased lung edema formation. Keywords: ALI, ARDS, histone, histone neutralization, STC3141, rat LPS model

Racial and Age-Related Differences in Impacts of High-Risk Cytogenetic Abnormalities on Survival in Multiple Myeloma in a Nationwide Electronic Health Record-Derived Database

Background: The incidence of multiple myeloma (MM) and enrichment of cytogenetic abnormalities differ significantly between racial/ethnic groups in the US, and their significance in determining myeloma progression and survival is not well understood. Whole genome sequencing has identified unique mutational signatures in MM, including an age-related process common in hyperdiploid myeloma. Our purpose was to describe racial and age-related differences in the impact of high-risk cytogenetic abnormalities (HRCAs) on survival in MM. Methods: We conducted a retrospective cohort study of adult MM patients starting first-line therapy between January 2011 and May 2021 using the nationwide Flatiron Health electronic health record-derived de-identified database. Patient-level demographic and clinical characteristics were ascertained using structured and unstructured data, curated via technology-enabled abstraction. Patients who had documented fluorescence in situ hybridization testing within 30 days prior to or 90 days following the start of first-line treatment were included. HRCAs, including gain or amplification 1q21, deletion 17p, t(4;14), t(14;16) and t(14;20), were identified and categorized as 0, 1, or 2+ HRCAs. Our outcomes of interest were real world progression free survival (rwPFS) and overall survival (rwOS). Cox proportional hazards models were used to calculate adjusted hazard ratios (HR) and 95% confidence intervals (CI), adjusted for demographic and clinical characteristics and treatment including time-dependent receipt of autologous stem cell transplantation. Results: From a cohort of 4889 MM patients, there were 790 (16%) Black and 2995 (61%) White patients with median ages at diagnosis of 68 and 70 years, respectively. Compared to White patients, a higher proportion of Black patients had IgG M-protein (61% vs 55%) and a lower proportion had 1+ HRCAs identified (31% vs 34%). Among all racial groups, compared to patients aged <65 years (N=1771), a higher proportion of patients aged 65+ years (N=3118) had IgA M-protein (21% vs 17%) and 1+ HRCAs identified (35% vs 33%). Multivariable models showed evidence of significant statistical interaction between age and prevalence of HRCA for rwPFS (P-int: 0.02). Among White patients, having 2+ HRCAs ("double-hit MM") compared to no HRCAs was associated with worse rwPFS in both younger and older patients (<65 years: HR 2.88, 95% CI 1.93-4.32, P<0.01; 65+ years: HR 1.51, 95% CI 1.18-1.94, P<0.01). Among Black patients, associations between double-hit MM and rwPFS were attenuated and not statistically significant regardless of age (<65 years: HR 1.81, 95% CI 0.69-4.74, P=0.23; 65+ years: HR 1.61, 95% CI 0.92-2.81, P=0.09). Similarly, we also found evidence of statistical interaction between age and prevalence of HRCA for rwOS (P-int: 0.02). Among White patients, double-hit MM was significantly associated with worse rwOS but the magnitude of increased risk differed for younger (HR 3.39, 95% CI 2.24-5.14, P<0.01) and older (HR 1.61, 95% CI 1.27-2.05, P<0.01) patients. Double-hit MM was significantly associated with worse rwOS among older Black patients (HR 1.78, 95% CI 1.03-3.06, P=0.04), but not younger Black patients (HR 1.60, 95% CI 0.58-4.40, P=0.36). Conclusions: In this cohort of newly diagnosed MM patients treated in routine practice, having double-hit MM was differentially predictive of poor survival across age groups. Double-hit MM was associated with worse rwPFS and rwOS among White patients, but these trends were less consistent among Black patients. Our current understanding of cytogenetic risk stratification of MM requires further study and additional data for identifying low- and high-risk subsets of patients across different ages and racial groups. Figure. Kaplan-Meier survivor functions for rwPFS in White (Panel A) and Black (Panel B) patients by age group and number of HRCAs Figure 1 Figure 1. Disclosures Calip: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company; Pfizer: Research Funding. Ascha: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company. Wang: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Pierre: Flatiron Health, Inc: Current Employment; Roche: Current holder of stock options in a privately-held company. Maignan: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company. Wadé: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Leng: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Seymour: Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Current equity holder in publicly-traded company; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Flatiron Health Inc: Current Employment. Patel: Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Neparidze: Eidos Therapeutics: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Research Funding; Janssen: Research Funding.

Btm-3566, a Novel Activator of the Mitochondrial Stress Response Promotes Robust Therapeutic Responses in Vitro and In Vivo in Diffuse Large B-Cell Lymphoma

Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or patients who fail CAR-T-cell therapy. r/r-DLBCLs are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class compound with activity against a variety of B-cell malignancies but with greatest effect in DLBCL. BTM-3566 activates the mitochondrial integrated stress response (ISR) through a novel mechanism regulated by the mitochondrial protein FAM210B. BTM-3566 induces apoptosis in DLBCL lines in vitro and complete tumor regression in vivo in DLBCL PDX mouse models harboring genetic alterations associated with poor prognosis. BTM-3566 is an oral small molecule based on a pyrazolothiazol-backbone, developed for treatment of diffuse large B-cell lymphoma (DLBCL). BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC 50 of ~200 - 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma lines. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% of oral bioavailability and close to 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. BTM-3566 showed stability in human hepatocytes (IC < 5 ml/min*kg) as well and a favorable in vitro safety profile. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete regression in all tumor-bearing animals. Most importantly, no subsequent tumor growth occurred for 2 weeks after cessation of therapy, indicating that treatment with BTM-3566 resulted in a durable complete remission in this model of double-hit DLBCL. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, including Myd88 mutations and MYC and BCL2 rearrangements, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1). Transcriptome and proteome analyses revealed that BTM-3566 strongly activated the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequent upregulation of the transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined, using CRISPR-Cas 9 gene depletion, that HRI was uniquely required for BTM-3566 eIF2a phosphorylation, induction of ATF4 ISR and apoptosis. HRI is described as being activated by mitochondrial-related stress, including heme depletion, increased ROS generation or blockage of mitochondrial ATP synthesis which result in an increase in mitochondrial proteostasis including activation of mitochondrial protease OMA1. We determined that BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Treatment with BTM-3566 induced OMA1-dependent OPA1 processing and mitochondrial fragmentation in as little as 30 minutes of treatment, in the absence of any reduction in mitochondrial oxygen consumption or membrane depolarization. This data indicates that BTM-3566 represents a new class of compounds that activate the mitochondrial protease OMA1. Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a mitochondrial membrane protein, negatively correlated with response to BTM-3566. Notably, overexpression of FAM210B completely prevents OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis in DLBCL cell line BJAB and the Burkitt lymphoma cell line Ramos. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation. In summary, we describe here a novel, highly potent activator of the mitochondrial ISR, which is well tolerated in mice and dogs, has favorable pharmacokinetics and induces robust DLBCL regression in-vivo. An IND application in B-cell malignancies will be completed by early Q1 2022 with initiation of first in human clinical trials the first half of 2022. Figure 1 Figure 1. Disclosures Schwarzer: Bantam Pharmaceutical: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anantha: Bantam Pharmaceutical: Consultancy, Current Employment, Current holder of stock options in a privately-held company. Cooper: Bantam Pharmaceutical: Consultancy. Hannink: Bantam Pharmaceutical: Research Funding. Hembrough: Bantam Pharmaceutical: Consultancy. Levine: Bantam Pharmaceutical: Consultancy, Current holder of individual stocks in a privately-held company. Luther: Bantam Pharmaceutical: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Stocum: Bantam Pharmaceutical: Current Employment, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Liesa-Roig: Bantam Pharmaceutical: Research Funding. Kostura: Bantam Pharmaceutical: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties: Named Inventor on patents with assignment to Bantam Pharmaceutical.

Early Mortality and Aggressive Presentation Lead to Poor Outcomes in Patients with Newly Diagnosed Double Hit and Triple Hit Multiple Myeloma

INTRODUCTION mSMART classifies high-risk Multiple Myeloma patients into Double Hit and Triple Hit Myeloma (DH/THM) on the basis of the number of high-risk cytogenetic abnormalities detected. The outcome of these patients with the use of proteasome inhibitors and IMiDs with upfront Auto HSCT is not known in the real-world setting. We retrospectively studied the outcome of these patients in a resource constrained setting. METHODS The case records of all newly diagnosed multiple myeloma patients who fulfilled the criteria for active myeloma between January 2018 and December 2020 were identified. The diagnosis of double hit and triple hit myeloma was based on mSMART classification (Presence of two or more of the following: IgH-FGFR3 translocation, IgH-MAF translocation, TP53 deletion, gain of chromosome 1q). Their case records were retrieved and information regarding baseline characteristics, therapy and outcomes was noted. RESULTS A total of 55 patients with newly diagnosed DH/THM were treated at our center during the study period. Median age of the cohort was 60 years with almost an equal number of male and female patients (M= 26; F= 29). Renal failure (serum creatinine >2.0mg/dl) was present in 33 patients (60%) while bone lesions, anemia (Hemoglobin <10 gm/dl) and hypercalcemia (serum calcium >12mg/dl) were present in 44 (80%), 51 (92.7%) and 24 (43.6%) patients respectively. Six patients (10.9%) fulfilled the criteria for plasma cell leukemia. All but 2 patients had gain of 1q with at least 3 copies. The most common cytogenetic combination was IgH-FGFR3 translocation with gain of 1q, which was seen in 28 patients (50.9%). This was followed by co-occurrence of TP53 deletion with gain of chromosome 1q in 11 patients (20%). Nine patients (16.4%) had triple hit myeloma. Bortezomib was used in the initial therapy of 43 patients (78.2%) and IMiDs (lenalidomide, thalidomide and pomalidomide) were used in the initial therapy of 32 patients (58.2%). Most patients received triplet therapy (N=32; 58.2%) with the most common regimen being RVd. Nine patients (16.4%) died within the first month of diagnosis and another 7 died in next month [15 patients in 2 months (29.1%)]. The most common cause of death within 2 months was progressive disease (N=13; 81.3%). Twenty-two patients (40%) achieved VGPR or better with anti-myeloma therapy and 7 patients (12.7%) underwent autologous HCT. Of the 39 evaluable patients, 21 patients (53.8%) relapsed during follow up with a median EFS of 8 months. Of the 7 patients who underwent transplant, 5 patients have had a follow-up of more than 1 year, of whom 3 have relapsed. One patient with post-transplant relapse died with progressive disease and CMV colitis. Median follow up of the entire cohort was 11 months (Range- 0 to 35 months). After excluding patients who died within the first 2 months of diagnosis, the median follow up was 14 months (Range- 3 to 35 months). Thirty-three patients (60%) patients died during follow-up. The most common cause of death was active or progressive disease (25 patients, 78.1%). Median OS for the cohort was 13 months. On univariate analysis, survival was better for patients without renal injury at presentation (29 months vs 6 months; p=0.007) and patients with <5% circulating plasma cells (14 months vs 2 months; p=0.045). Patients who achieved VGPR had a better OS than patients who did not (Not reached vs 3 months; p=0.000) as did patients who underwent auto transplant (Not reached vs 10 months; p=0.023). OS did not significantly differ with TP53 deletion status, or number of copies of 1q (3 or greater than 3). In the multi-variate analysis, presence of renal failure (Hazard ratio- 2.663; p-0.017) and >5% circulating plasma cells (Hazard ratio- 3.082; p-0.020) were significantly associated with increased risk of mortality, while achievement of VGPR or better with therapy was associated with longer survival (Hazard ratio- 0.174; p-0.001). DISCUSSION The outcome of DH/THM remains poor in the real-world setting. The outcome was not affected by specific high-risk cytogenetic abnormalities or their combination in our study. Progressive disease within the first two months of diagnosis was the most common cause of death in more than 1/3 rd of the patients. Novel therapies and protocols are required to improve outcomes for this group of multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.