Jeffrey W. Craig

CD24 is highly expressed in aggressive B-cell malignancies and represents a novel immunologic vulnerability to overcome resistance to CD19 chimeric antigen receptor T-cell (CAR-T) therapy. We developed a CD24 × CD3 bispecific antibody (CD24 BsAb) and CXCR3-armed CD24 CAR-T cells, both exhibiting potent antitumor activity in lymphoma and leukemia models. CD24 BsAb and CD24 CAR-T overcome CD19 CAR-T resistance driven by antigen loss and apoptosis defects in vitro and in vivo. Beyond direct cytotoxicity, CD24-targeted T cells reprogram macrophages toward an M1-like phenotype and enhance phagocytic clearance. We further engineered a dual-specific, dual-functional CD19 CAR-T that secretes a CD24 bispecific T-cell engager (CD24 BiTE), enabling simultaneous targeting of CD19 and CD24 to address tumor heterogeneity and achieve superior efficacy. Collectively, our study demonstrates that CD24-directed adoptive T-cell therapies are warranted for B-cell malignancies through robust induction of both innate and adaptive immune responses.

Diffuse large B-cell lymphoma (DLBCL) is a biologically heterogeneous disease. Two genomic classification systems, LymphGen and DLBclass, are capable of reproducibly classifying single tumors into subtypes with prognostic relevance in the context of standard-of-care R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) chemoimmunotherapy. Preliminary data from subgroup analyses of clinical trials suggest that the distinct biological features are targetable with drugs that exploit subtype-specific vulnerabilities, and large clinical trials are being designed to test this hypothesis. Although whole-exome sequencing (WES) remains the gold standard for each of these classification systems, smaller targeted sequencing panels reduce costs associated with sequencing, data storage, and processing. Here, we describe the design and validation of a targeted sequencing panel (LySeqST) that captures the genomic features required specifically for LymphGen classification. We perform in silico theoretical and real-world validation vs WES data and demonstrate that LySeqST can be used to accurately classify DLBCL tumors using both LymphGen and DLBclass. In an unselected population-based cohort, we determine the real-world proportions and validate the prognostic relevance of LymphGen subtypes, confirming that only tumors expressing the dark zone gene expression signature are consistently associated with inferior outcomes. Our results support the use of LySeqST for accurate genomic classification of DLBCL.

The myeloid transcription factors PU.1 and CCAAT/enhancer-binding protein α (C/EBPα) are essential for monocyte/macrophage (Mo/MΦ) development, and their dysregulation has been linked to myeloid malignancies and immune disorders. Although their binding to enhancers for myeloid coding genes is established, their control of noncoding regulatory RNAs remains poorly understood. Using a comprehensive collection of putative and verified enhancers, we profiled the PU.1 cistrome and transcriptome, identifying a subset of noncoding genes that are both associated with PU.1-bound enhancers and regulated by PU.1. Notably, PU.1 induces expression of LOUP, an enhancer RNA transcribed from a locus containing a conserved PU.1 cis-regulatory element cluster, which is characterized by features of myeloid-specific enhancers. Disruption of a PU.1-binding motif in the LOUP promoter and the enhancer reduced LOUP promoter activity, whereas mutation of another PU.1-binding site within the LOUP gene body and the enhancer, which modulates enhancer-promoter interaction, diminished both Pu.1 and Loup levels in mice. The myeloid transcription factor C/EBPα, which binds to the enhancer, is necessary for PU.1 and LOUP expression as inducible deletion of Cebpa in mice led to their downregulation. LOUP depletion impaired Mo/MΦ marker and inflammatory cytokine expression as well as phagocytic function. Collectively, our findings reveal that PU.1 and the enhancer RNA LOUP form a previously unrecognized feed-forward loop, induced by C/EBPα, that drives their mutual expression and establishes a regulatory circuit. This circuit programs monocyte to macrophage differentiation and innate immune function, providing important implications for inflammatory diseases and myeloid malignancies.

Aggressive B-cell lymphomas, driven by MYC overexpression, exhibit rapid progression, resistance to therapies, and poor survival. While chimeric antigen receptor (CAR)-engineered T cells have demonstrated remarkable clinical efficacy in B-cell lymphomas, nearly half of patients who initially respond to CAR-T therapy eventually develop resistance and disease progression. In this study, we report the presence of residual drug-tolerant persister (DTP) and resistant lymphoma cells remaining within a highly immunogenic tumor microenvironment (TME) induced by the MCL-1 inhibitor (MCL-1i) S63845. MCL-1 inhibition downregulates MYC and activates the STAT1-interferon inflammatory pathway, promoting cytotoxic T-cell infiltration with reduced tumor-associated myeloid cells both in vitro and in vivo. Sublethal dose of the MCL-1i enhances TME immunogenicity and reawakens anti-tumor immune responses in murine models. We show that MCL-1i and CD19-targeted CAR-T cells reciprocally overcome resistance to each single-agent therapy, and combining MCL-1i with CD19 CAR-T cells significantly improves treatment efficacy, resulting in near-complete eradication of MYC-driven lymphoma in vivo. Together, these findings highlight a synergistic, dual-pronged therapeutic strategy targeting both tumor-intrinsic survival pathways and the immunosuppressive TME. This combinatorial one-two-punch approach offers a promising path to eliminate DTP and residual disease, prevent relapse and pave the way for deep clinical remissions in aggressive B-cell lymphomas.

Patients with diffuse large B-cell lymphoma (DLBCL) with late relapse (>2 years from diagnosis) may be treated with a second course of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone)–like therapy particularly if comorbidities preclude more intensive options. Patients with de novo DLBCL initially treated with an R-CHOP–like regimen who later developed late relapse and were re-treated with curative-intent R-CHOP–like therapy were identified using the BC Cancer databases. Sixty-five patients were identified; at relapse, the median age was 77 years (range, 52-89), 81% had stage III to IV DLBCL, 52% had an Eastern Cooperative Oncology Group performance status of 2 to 4, and 78% had an International Prognostic Index score of 3 to 5. The median time from original diagnosis was 7.4 years (range, 2.5-15.9). Median number of cycles of R-CHOP–like therapy received at relapse was 5 (range, 1-6). Overall response rate was 72%, and 57% complete response. With a median follow-up of 31 months, 2-year time to progression (TTP) was 54%, 2-year progression-free survival was 46%, 2-year disease-specific survival was 64%, and 2-year overall survival was 54%. Patients relapsing >5 years from diagnosis had better TTP, with a 2-year TTP of 66% compared with 9% for patients relapsing between 2 and 5 years (hazard ratio, 0.30; 95% confidence interval, 0.14-0.64; P = .001). Many patients with late-relapsing DLBCL may be effectively treated with further R-CHOP–like therapy, avoiding more intensive and costly secondary therapies.

High-grade B-cell lymphoma with "double-hit" MYC and BCL2 rearrangements (HGBCL-DH-BCL2) is associated with poor outcomes following standard chemoimmunotherapy, prompting dose-intensive regimen use. However, the benefit of intensification is unclear due to rarity precluding randomized trials, and selection bias in retrospective comparisons. In 2015, BC Cancer introduced a provincial guideline recommending dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R) for fit patients aged ≤75 years with HGBCL-DH-BCL2 identified by routine cytogenetic testing. To assess this guideline’s impact, we compared the outcomes of patients with de novo HGBCL-DH-BCL2 tumors of diffuse large B-cell lymphoma (DLBCL) morphology across 2 eras. The DA-EPOCH-R era (2015-2020) included patients diagnosed after guideline implementation. The historic era (2005-2010) included patients identified from a historic province-wide cohort of patients with DLBCL morphology tumors that underwent universal cytogenetic testing in a research setting, predominantly treated with standard chemoimmunotherapy. Two-year overall survival (OS) was significantly improved in the DA-EPOCH-R vs historic era (75% vs 47%, P = .008) in HGBCL-DH-BCL2, whereas OS remained unchanged in DLBCL, not otherwise specified (78% vs 76%, P = .17). Within HGBCL-DH-BCL2, tumors harboring immunoglobulin MYC partner loci (43%) and those expressing the dark-zone signature (77%) were associated with the most substantial survival improvements. In a contemporary cohort of HGBCL-DH-BCL2 histologically transformed from follicular lymphoma (FL) in the DA-EPOCH-R era, outcomes of patients who were chemoimmunotherapy-naïve were comparable to those with de novo disease, whereas patients treated with chemoimmunotherapy for FL prior to transformation had poor outcomes. These data support using DA-EPOCH-R in select patients with HGBCL-DH-BCL2 of DLBCL morphology.

Histone deacetylase inhibitors (HDACis) are valued treatment options for patients with T-cell malignancies. Romidepsin is a selective class I HDACi initially approved for patients with relapsed or refractory cutaneous and peripheral T-cell lymphomas (PTCLs). Romidepsin was withdrawn from its PTCL indication following a negative randomized phase 4 study (romidepsin-CHOP [cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone]) that showed no benefit over CHOP alone, further diminishing options for patients. Herein, we describe the development of, to our knowledge, a first-in-class polymer nanoparticle (PNP) of romidepsin using an innovative amphiphilic diblock copolymer–based nanochemistry platform. Nanoromidepsin exhibited superior pharmacologic properties with improved tolerability and safety in murine models of T-cell lymphoma (TCL). The PNP also exhibited superior antitumor efficacy in multiple models, including in vitro TCL cell lines, ex vivo samples from patients with large granular lymphocyte (LGL) leukemia, and murine TCL xenografts. Nanoromidepsin demonstrated greater accumulation in tumors and a statistically significant improvement in overall survival compared with romidepsin in murine xenograft models. These findings justify the clinical development of nanoromidepsin in patients with T-cell malignancies.

Background: 18-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) scans have largely replaced bone marrow biopsy in the staging workup for diffuse large B-cell lymphoma (DLBCL). Purpose/Methods: Here, we highlight a pitfall of FDG PET/CT for assessing bone marrow involvement, detailing the clinical course of a patient diagnosed with otherwise limited-stage DLBCL who exhibited diffuse bone marrow FDG uptake, felt to be compatible with extensive lymphomatous infiltration. In accordance with current National Comprehensive Cancer Network guidelines, this finding circumvented the need for a formal staging bone marrow biopsy procedure. Results: Subsequent clinical developments revealed extensive bone marrow involvement by a second, unexpected malignancy (multiple myeloma), casting doubt on the assumption that the initial diffuse marrow uptake was attributable to DLBCL. Conclusion: The presented case emphasizes the challenges of interpreting diffuse FDG uptake within the bone marrow compartment and highlights the inherent contribution of histomorphology for the accurate diagnosis and classification of hematolymphoid neoplasms.

Over-expression of BCL-2 defines follicular lymphoma (FL). Venetoclax (VEN), a selective BCL-2 inhibitor, has previously been evaluated with bendamustine-based chemoimmunotherapy. VEN was given continuously, resulting in promising efficacy but unacceptable toxicity. The Phase II PrE0403 study was designed to evaluate intermittent dosing of VEN (10 days per cycle) combined with obinutuzumab and bendamustine (VEN-OB) in untreated FL subjects with high-risk features defined as a FLIPI-1 score of ≥3 and/or high tumor burden by GELF criteria. A total of 56 subjects were planned to be accrued with a goal of having 51 subjects eligible to improve the historical 50% CR rate to 65% with an 85% power and 15% type I error rate. Immunohistochemistry (IHC) expression of 3 antiapoptotic proteins (BCL-xL, MCL-1, and BCL-2) was performed and correlated with clinical outcomes. All 56 subjects were eligible and treated. CR rate was 41/56 (73.2%) and ORR was 52/56 (92.5%) meeting the primary endpoint. 2-year estimated PFS was 87.5% (90% CI: 75.3,93.9%) and 2-year estimated OS was 94.6% (90% CI: 86.7, 97.9%). However, the incidence of treatment-related adverse events ≥ grade 3 was 83.9% and serious adverse events were seen in 57.1%. After induction, atypical infections, including Grade 5 events, occurred. Anti-apoptotic protein expression by IHC was not correlated with clinical outcomes. Thus, while meeting the primary efficacy end point, VEN-OB is considered overly toxic in high-risk FL.

High-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2), or “double-hit lymphoma,” has been associated with a high risk of central nervous system (CNS) relapse. However, historic estimates are impacted by selection bias. We report CNS relapse rates associated with HGBCL-DH-BCL2 from a population-based cohort with complete fluorescence in situ hybridization testing, as well as diffuse large B-cell lymphoma morphology (DLBCL) tumors expressing the dark-zone gene expression signature (DZsig), which was originally derived from HGBCL-DH-BCL2. The 2-year CNS relapse risk in HGBCL-DH-BCL2 was 6.8%. CNS relapses were early, predominantly leptomeningeal (73%), and co-occurred with systemic relapse (64%). High-risk CNS International Prognostic Index (CNS-IPI) and concordant bone marrow involvement were associated with an elevated CNS relapse risk in HGBCL-DH-BCL2. The “refined cell-of-origin” classification assigned 20% of DLBCL morphology tumors with germinal center B-cell–like phenotype (GCB-DLBCL) into a distinct subgroup based on DZsig expression (DZsig+). CNS relapse risk in DZsig+ (2 year: 6.4%) was independent of HGBCL-DH-BCL2 status and was further stratified by the CNS-IPI. CNS relapse in DZsig-negative GCB-DLBCL was rare (2-year risk, 1.4%; P = .04 vs DZsig+) and exclusively parenchymal. Altogether, the CNS relapse risk in HGBCL-DH-BCL2 is lower than previously reported, and DZsig refines risk stratification in GCB-DLBCL.