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Blood Podcast
“Ironing out” Tet2-mutant HSPCs; A CAR-T “license to kill” in T cell leukemia/lymphoma; insights on cHL genetics, through the lens of ctDNA
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Welcome to the September 4, 2025 episode of Blood Podcast, your source for innovative ideas and cutting edge information. Our topics are based on articles published in Blood, A Journal of the American society of hematology. First on the podcast Tet2 is often mutated in myeloid malignancies and clonal hematopoiesis. In new work, expansion of Tet2 mutant HSPCs was dependent on NCOA4, the cargo receptor. Mediating ferritinophagy will iron out the implications after that a 007 license to kill in T cell leukemia lymphoma. Wu car T 007 is an off the shelf CAR T product with manageable safety and encouraging efficacy. With further work, it could become a new option for patients in urgent need of therapy. Finally, a comprehensive genetic study of classical Hodgkin lymphoma using circulating tumor DNA. This new research provides novel and complex insights on genetic subtypes, prognostic biomarkers, neoantigens in the disease environment and more. First, let's unpack the link between NCOA4 mediated ferritinophagy and the expansion of Tet2 mutant HSPCs, hematopoietic stem and progenitor cells. This comes from an article in Blood titled An in vivo barcoded CRISPR Cas9 screen identifies NCOA4 mediated ferritinophagy as a dependence in Tet2 deficient hematopoiesis. The first author is Justin Loke, affiliated with Dana Farber Cancer Institute in Boston and the University of Birmingham in the United Kingdom. This story starts with Tet 2, an iron dependent enzyme that catalyzes a process that is crucial in DNA demethylation. We know that loss of function mutations in Tet 2 are common in CHIP clonal hematopoiesis of indeterminate potential and in myeloid malignancies, including acute myeloid leukemia and myelodysplastic syndromes. And we know that Tet 2 deficient HSPCs have a clonal advantage. They expand more rapidly than wild type HSPCs in mouse models, Tet2 loss increases the capacity of HSPCs for self renewal and repopulation over the long term. However, the why behind this clonal advantage is not as clear. As such, the work of Lok and co authors is intriguing and they identify a selective dependency in Tet2 deficient cells, one that could have broad translational significance. That dependency is related to NCOA 4, which targets ferritin for lysosomal degradation. However, this story is not just about an isolated dependency. It's also about the technology that made the identification of this dependency possible. The use of genetic screens is one potential strategy to identify selective dependencies in somatically mutated cells. Through in vivo genetic screens, it's possible to access aspects of normal and neoplastic biology that exist only within the native microenvironment of hscs hematopoietic stem cells. Yet there is one major limitation to genetic cellular heterogeneity, the tremendous diversity of differentiation and proliferative potential of HSPCs. To address that challenge, Loke et al. Developed a large scale lentiviral CRISPR Cas9 screening platform, one that leverages a so called barcoding strategy. Through this platform, researchers can induce gene perturbations and clonally track their growth over long term transplantation using Barcodes nucleotide based unique molecular identifiers3. Through barcoding, authors say they can identify true genetic dependencies by tracking mutant clones with late clonal growth. Loke and colleagues used this CRISPR Cas9 platform to identify vulnerabilities in a model of Tet2 knockout HSPC expansion in mice. As background, conditional knockout modeling of Tet 2 faithfully recapitulates the clonal expansion observed in clonal hematopoiesis, including an increase in HSC renewal and premalignant state. The key finding is that the authors pinpointed NCOA4 as a selective genetic vulnerability in Tet2 knockout HSPCs. In their screen, NCOA4 was selectively required for Tet2 knockout clonal outgrowth as compared to wild type. You may recall that NCOA 4 is an adapter protein that targets ferritin for lysosomal degradation. NCOA4 directs ferritin to lysosomes resulting in ferritinophagy. In other words, autophagy dependent degradation of ferritin. In the present work, Loc et al Also identify labile iron as a dependency for the competitive advantage of Tet2 knockout HSPCs identified through previous research. They also show that in Tet2 mutant stem progenitor cells, NCOA4 mediated ferritinophagy maintains iron availability for increased mitochondrial production of ATP. The Tet 2 deficient HSPCs had increased mitochondrial demand in comparison to the wild type cells. Authors explain that in Tet2 deficient HSPCs, increased mitochondrial ATP production correlates with increased cellular iron requirements and in turn promotes NCOA4 dependent ferritinophagy. Their data suggest that the knockout cells have increased ferritinophagic flux and an expanded labile iron pool and the release of labile iron in response to cellular demands maintains intracellular iron homeostasis. Interestingly, the authors were able to inhibit ferritinophagy first through genetic deletion of NCOA4. This impaired the long term fitness of tattoo deficient cells without affecting wild type HSPCs. They also blocked ferritinophagy using ironomycin, a lysosome targeting compound. Ironomycin sequesters iron inhibiting its lysosomal release. In vivo treatment with ironomycin selectively impaired the expansion of tattoo knockout HSPCs with a marked effect on the myeloid lineage. Thus, by targeting ferritinophagy, it may be possible to iron out our problems with tattoo mutant cells. That pun comes courtesy of Clement Leroux and Jerome Tamberini, authors of a commentary exploring the implications of these findings. Leroux is with the University of Toulouse in France and Tamberini hails from the University of Geneva in Switzerland. According to Leroux and Tamberini, this work identifies ferritinophagy as a critical metabolic dependency in tet deficient clonal hematopoiesis. They say these findings are consistent with recent reports identifying ferritinophagy as a critical dependency of leukemic stem cells in patient derived xenograft models of acute myeloid leukemia. As such, NCOA4 mediated iron mobilization may be a common metabolic feature of both pre leukemia leukemic and leukemic stem cells. Together with other recent studies, this work highlights enhanced mitochondrial metabolism as a hallmark of clonal hematopoiesis. It also suggests that metabolic vulnerabilities may be therapeutically exploitable to prevent or delay malignant progression as the field of clonal hematopoiesis continues to expand. COMMENTARY Authors conclude studies such as this will be key in the translation of molecular discoveries into interventions that could alter the trajectory of disease in its earliest ST. Next is a phase 1. 2 trial of anti CD7 allogeneic who car T007 in patients with relapsed refractory T cell malignancies. The first author is Armin Gobatti of the Washington University School of Medicine in St. Louis, Missouri. Gobatti & Co authors describe a promising new treatment for patients with relapsed or refractory T cell acute lymphoblastic leukemia and lymphoma. This is an area of substantial unmet medical need. Patients with relapsed or refractory disease generally have a dismal prognosis and few effective treatment options. Median survival is roughly six months and five year survival is less than 7%. Since 2005 only one drug has received regulatory approval, Nilarabine, which had response rates just over 20% and was associated with dose limiting neurotoxicity. Now Gobody et al describe a CAR T cell therapy that targets CD7. This T cell antigen is expressed in more than 95% of T cell acute lymphoblastic leukemias and lymphomas. In both adult and pediatric cases, CD7 is expressed consistently at diagnosis, relapse and minimal residual disease target antigen aside, those who know the history of CAR T cell developments may be skeptical. In B cell malignancies, CAR T cell products have been revolutionary, but T cell malignancies have been challenging. One issue is the potential for malignant cells to contaminate the CAR T cell product. Another concern is fratricide, the self killing of T cells due to shared expression of target antigens on T effector cells and T cell malignancies. However, Wu Car T007 has the potential to overcome both challenges. This off the shelf allogeneic CAR T cell product was engineered to be fratricide resistant. WUCAR T007 is built on T cells generated from healthy donors. Using this allogeneic cell source avoids the risk of malignant cell contamination. Those T cells are edited using CRISPR Cas9 to delete CD7, mitigating the risk of fratricide. The cells are then Transduced with a second generation chimeric antigen receptor targeting CD7 expanded, purified and cryopreserved. Now Gobadi et al report the primary analysis of a global multicenter study assessing the safety and efficacy of WCAR T0007 in patients 12 years of age or older. Patients received standard or enhanced lymphodepleting chemotherapy followed by one infusion of Wucart 007. The study included a phase one dose escalation component followed by a phase two expansion cohort in which the recommended phase two dose was 900 million cells with enhanced lymphodepletion. Investigators enrolled patients with relapsed refractory T cell acute lymphoblastic leukemias and lymphomas. Patients had received a median of four lines of previous therapy. Approximately 40% had relapsed following a previous allogeneic HSCT hematopoietic STEM cell transplant. Thirteen received the recommended phase 2 dose in the expansion cohort. Among the 11 patients evaluable for response, the overall response rate was 91%. The composite rate of CR complete remission defined as CR CR with incomplete hematologic recovery was 73%. The single infusion of WuCart 007 was associated with acceptable toxicity. The most common treatment related adverse event was cytokine release syndrome occurring in 88.5% of patients 19.2% were grade 3 to 4 in severity. Immune effector cell associated neurotoxicity syndrome events occurred in two patients or 7.7%. Both were grade one. Investigators also reported one grade two acute graft versus host disease event, N1 grade two immune effect or cell associated HLH like syndrome. In a commentary on this study, authors take a cue from James Bond. They titled their commentary car T007A license to Kill. The authors are Swati Naik of St. Jude Children's Research Hospital in Memphis, Tennessee and Maxime Momonkin of Baylor College of Medicine in Houston, Texas. Naik and Momonkin say that despite the relatively small numbers reported, this study offers the promise of a new treatment option for patients with otherwise dismal outcomes. They describe the overall response rates as encouraging in this high risk cohort of patients. Eight patients transitioned to HSCT after WUCART 007 and had persistent CRS, suggesting the potential of this product to serve as a successful bridge to transplant and as an off the shelf product. Wucart 007 can address manufacturing challenges associated with autologous CAR T, offering patients an immediate therapeutic option. However, Commentary authors add there are also limitations to an off the shelf approach. Immune rejection of infused cells shortens their functional persistence and consolidative therapies are needed to maintain responses. WUCAR T007 had limited persistence, with expansion bolstered by use of enhanced lymphodepletion. On the other hand, the limited duration of WUCAR T007 activity allowed for a more rapid rebound of endogenous CD7T and NK cells. To date, several different CD7 car T strategies for T cell malignancies have been reported, autologous donor derived and off the shelf. According to Commentary authors, the current data indicate that an off the shelf approach can be an effective bridge to hsct, providing an option for patients in urgent need of therapy or with low T cell counts. Future efforts, they conclude, should be focused on optimizing safety and therapeutic sequencing, which could hold promise for long term cures. Finally, on today's podcast, a comprehensive genetic study of classical Hodgkin lymphoma using circulating tumor DNA or ctdna. The first author is Maria Cristina Perosa of the Institute of Oncology Research in Bellinzona, Switzerland Perosa and co authors report novel insights made possible by CTDNA analysis, which addresses a major hurdle in prior genetic studies. CHL tumors yield a very limited number of Hodgkin Reed Sternberg cells for analysis. The malignant cells make up as little as 0.1% of the tumor. To overcome this technical hurdle, tumor cells need to be enriched. However, CTDNA is changing the equation. Plasma from a CHL patient contains a higher concentration of neoplastic DNA as compared to the bulk tumor. This increases the appeal of CTDNA analysis as a reliable method for molecular profiling. In the present study, Pirosa et al analyzed a large cohort of CHL patients focused on the coding regions of 155genes recurrently mutated in mature B cell malignancies. They also studied 33 regulatory non coding regions impacted by AID mediated somatic hypermutation, that's AED as in activation induced cytodyne deaminase. Their investigations revealed two distinct genetic subtypes of Chl. The first, which encompassed 64% of cases, was characterized by a higher mutational load plus a higher fraction of mutations associated with AID and microsatellite instability. The second, accounting for 36%, was characterized by chromosomal instability and a higher level of somatic copy number alterations. These investigations also revealed insights on genetics in relation to outcomes. Whole genome duplication was more common in CHL compared to other B cell malignancies, and detection of WGD in CTDNA was associated with a shorter progression free survival and with a lower chance of cure following initial treatment. Collectively, this evidence supports the use of WGD as a prognostic biomarker. Non coding regulatory mutations were highly prevalent. 83% of CHL tumors had DNA alterations in at least one of the non coding regulatory regions and many of the mutations showed AIDS signatures. Pirosa et al also identified a specific mutational hotspot in one non coding region. Approximately 30% of CHL cases had mutations involving BCl6, a transcriptional repressor involved in maintaining the germinal center phenotype in a variety of lymphomas after exposure to a potent BCL6 degrader core genes regulated by BCL6 were de repressed and proliferation was impaired. This indicates that BCL6 may be a potential vulnerability in a subset of cases. Further investigations provided insights into how neoantigens shape the CHL microenvironment. The number and clonality of neoantigens was associated with tumor microenvironment type based on gene expression profiling and immunohistochemistry. 2 Chl microenvironmental classes were macrophage enriched and T cell enriched. A higher load of neoantigens was detected in the T cell enriched class, suggesting that the composition of the CHL microenvironment is at least partially shaped by neoantigens. Finally, this report provides evidence that CTDNA evaluation of molecular response may improve the accuracy of PET ct, especially when PET CT results are ambiguous. PET CT scans can help guide treatment adjustments however, approximately 1/4 of results are false positives. Data from Pirosa et al. Indicate that in patients who show positive PET CT results, CTDNA may serve as a surrogate for tissue biopsy to verify tumor persistence. In conclusion, authors say that CTDNA analysis reveals genetic subtypes of CHL are driven more by mechanisms of genetic instability than by clustering of mutations into functional groups. Second, many factors including whole genome duplication, non coding BCL6 mutations and neoantigens are involved in shaping CHL pathophysiology and outcomes. These findings were reviewed by Andrej Havernek of Charles University in Prague, Czech Republic. In a commentary, Havernek says this new work provides novel and complex insights into CHL genetics. Pirosa et al. Show how various genetic events, such as novel mutations of the BCL6 locus, shape the CHL biology and treatment outcome. It's a comprehensive analysis of CHL genetics that shows for the first time AID mediated mutagenesis at non coding regulatory regions with substantial consequences on gene expression regulation in chl. It's a comprehensive analysis of CHL genetics that shows for the first time AID mediated mutagenesis at non coding regulatory regions with substantial consequences on gene expression regulation in chl. According to Haverneck, this discovery reveals an important important overlap of CHL and diffuse large B cell lymphoma biology. The current study clearly documents an important novel non coding regulatory mutational hotspot in the BCL6 gene. However, there could be other such hotspots contributing to the deregulated gene expression program of chl. The present study also supports the exploration of CTDNA as a clinical tool for monitoring treatment response with high CHL cure rates. Haverneck writes, Treatment personalization is one of the directions for further outcomes improvement. With better risk stratification and more reliable treatment response evaluation, therapy could be more precisely de escalated or intensified. However, robust validation is needed before CTDNA can be incorporated into routine clinical practice. Clinical trials are underway to evaluate use of CTDNA based chemotherapy adjustment and treatment monitoring. Haverneck concludes with an emphasis on developing technical standards for CTDNA assessment, which he says are needed at every level from the pre analytical phase to bioinformatics data processing to evaluation of results. You have been listening to the Blood Podcast. The articles mentioned in this podcast can be found@bloodjournal.org and are linked in the show notes of this episode. Be sure to join us next week for another episode. Thank you for listening.